Melanoma antigens and their use in diagnostic and therapeutic methods

ABSTRACT

The present invention provides a nucleic acid sequence encoding a melanoma antigen recognized by T lymphocytes, designated MART-1. This invention further relates to bioassays using the nucleic acid sequence, protein or antibodies of this invention to diagnose, assess or prognoses a mammal afflicted with melanoma or metastata melanoma. This invention also provides immunogenic peptides derived from the MRT-1 melanoma antigen and a second melanoma antigen designated gp100. This invention further provides immunogenic peptides derived from the MART-1 melanoma antigen or gp100 antigen which have been modified to enhance their immunogenicity. The proteins and peptides provided can serve as an immunogen or vaccine to prevent or treat melanoma.

This application is a divisional of application Ser. No. 09/898,860,filed Jul. 3, 2001, which is a divisional of application Ser. No.09/267,439, filed Mar. 12, 1999, now U.S. Pat. No. 6,270,778, which is adivisional of application Ser. No. 09/073,138, filed May 5, 1998, nowU.S. Pat. No. 6,537,560, which is a continuation-in-part of applicationSer. No. 08/417,174, filed Apr. 5, 1995, now U.S. Pat. No. 5,844,075,which is a continuation-in-part of application Ser. No. 08/231,565,filed Apr. 22, 1994, now U.S. Pat. No. 5,874,560, all of which arehereby incorporated by reference.

FIELD OF THE INVENTION

This invention is in the field of prevention and treatment of humancancers. More specifically, this invention relates to genes encodingmelanoma antigens recognized by T-Cells and their corresponding proteinsand to preventative, diagnostic and therapeutic applications whichemploy these genes or proteins.

BACKGROUND OF THE INVENTION

Melanomas are aggressive, frequently metastatic tumors derived fromeither melanocytes or melanocyte related nevus cells (“Cellular andMolecular Immunology” (1991) (eds) Abbas A. K., Lechtiman, A. H., Pober,J. S.; W. B. Saunders Company, Philadelphia: pages 340-341). Melanomasmake up approximately three percent of all skin cancers and theworldwide increase in melanoma is unsurpassed by any other neoplasm withthe exception of lung cancer in women (“Cellular and MolecularImmunology” (1991) (eds) Abbas, A. K., Lechtiman, A. H., Pober, J. S.;W.B. Saunders Company Philadelphia pages: 340-342; Kirkwood and Agarwala(1993) Principles and Practice of Oncology 7:1-16). Even when melanomais apparently localized to the skin, up to 30% of the patients willdevelop systemic metastasis and the majority will die (Kirkwood andAgarwala (1993) Principles and Practice of Oncology 7:1-16). Classicmodalities of treating melanoma include surgery, radiation andchemotherapy. In the past decade immunotherapy and gene therapy haveemerged as new and promising methods for treating melanoma.

T cells play an important role in tumor regression in most murine tumormodels. Tumor infiltrating lymphocytes (TIL) that recognize uniquecancer antigens can be isolated from many murine tumors. The adoptivetransfer of these TIL plus interleukin-2 can mediate the regression ofestablished lung and liver metastases (Rosenberg, S. A., et al., (1986)Science 233:1318-1321). In addition, the secretion of IFN-γ by injectedTIL significantly correlates with in vivo regression of murine tumorssuggesting activation of T-cells by the tumor antigens. (Barth, R. J.,et al., (1991) J. Exp. Med. 173:647-658). The known ability of tumor TILto mediate the regression of metastatic cancer in 35 to 40% of melanomapatients when adoptively transferred into patients with metastaticmelanoma attests to the clinical importance of the antigens recognized(Rosenberg, S. A., et al., (1988) N Engl J Med 319:1676-1680; RosenbergS. A. (1992) J. Clin. Oncol. 10:180-199).

T cell receptors on CD8³⁰ T cells recognize a complex consisting of anantigenic peptide (9-10 amino acids for HLA-A2), β-2 microglobulin andclass I major histocompatibility complex (MHC) heavy chain (HLA-A, B, C,in humans). Peptides generated by digestion of endogenously synthesizedproteins are transported into the endoplastic reticulum, bound to classI MHC heavy chain and β2 microglobulin, and finally expressed in thecell surface in the groove of the class I MHC molecule. Therefore, Tcells can detect molecules that originate from proteins inside cells, incontrast to antibodies that detect intact molecules expressed on thecell surface. Therefore, antigens recognized by T cells may be moreuseful than antigens recognized by antibodies.

Strong evidence that an immune response to cancer exists in human—isprovided by the existence of lymphocytes within melanoma deposits. Theselymphocytes, when isolated, are capable of recognizing specific tumorantigens on autologous and allogeneic melanomas in an MHC restrictedfashion. (Itoh, K. et al. (1986), Cancer Res. 46: 3011-3017; Muul, L.M., et al. (1987), J. Immunol. 138:989-995); Topalian, S. L., et al.,(1989) J. Immunol. 142: 3714-3725; Darrow, T. L., et al., (1989) J.Immunol. 142: 3329-3335; Hom, S. S., et al., (1991) J. Immunother.10:153-164; Kawakami, Y., et al., (1992) J. Immunol. 148: 638-643; Hom,S. S., et al., (1993) J. Immunother. 13:18-30; O'Neil, B. H., et al.,(1993) J. Immunol. 151: 1410-1418). TIL from patients with metastaticmelanoma recognize shared antigens including melanocyte-melanoma lineagespecific tissue antigens in vitro (Kawakami, Y., et al., (1993) J.Immunother. 14: 88-93; Anichini, A. et al., (1993) et al., J. Exp. Med.177: 989-998). Anti-melanoma T cells appear to be enriched in TILprobably as a consequence of clonal expansion and accumulation at thetumor site in vivo (Sensi, M., et al., (1993) J. Exp. Med.178:1231-1246). The fact that many melanoma patients mount cellular andhumoral responses against these tumors and that melanomas express bothMHC antigens and tumor associated antigens (TAA) suggests thatidentification and characterization of additional melanoma antigens willbe important for immunotherapy of patients with melanoma.

Peripheral blood lymphocytes have been used to identify potentialmelanoma tumor antigens. Van Der Bruggen et al. (1991) Science 254:1643-1647 has characterized a gene coding for a melanoma antigen,designated MAGE-1, using T cell clones established from the peripheralblood of patients who were repetitively immunized in vivo withmutagenized tumor cells. Cytotoxic T-cells derived from the peripheralblood lymphocytes of patients with melanoma were used to identify apotential antigenic peptide encoding MAGE-1 (Traversari, C., et al.(1992) J. Exp. Med. 176:1453-1457). Brichard et al. (1993) J. Exp. Med.178:489-495 has also characterized a gene encoding a melanoma antigendesignated tyrosinase using peripheral blood lymphocytes from patientswho were sensitized by repetitive in vitro stimulation with tumor.Further support for the therapeutic potential of melanoma antigens isprovided by Brown et al. (U.S. Pat. No. 5,262,177). Brown et al. (U.S.Pat. No. 5,262,177) relates to a recombinant vaccinia virus-basedmelanoma vaccine where the melanoma antigen p97 is reported to show aprotective effect from tumor cell challenge in a murine model.Characterization of additional melanoma antigens is important for thedevelopment of new strategies for cancer immunotherapy, in particularfor melanoma.

SUMMARY OF THE INVENTION

This invention relates, in general, to nucleic acid sequences encodingmelanoma antigens recognized by T-lymphocytes (MART-1) and protein andpeptides encoded by these sequences. This invention further providesbioassays for these nucleic acid sequences, proteins and peptides. Thisinvention also provides peptides which have been derived from the MART-1amino acid sequence and modified to enhance their immunogenocity. Thisinvention also provides therapeutic uses for the nucleic acid sequences,proteins, peptides or modified peptides described herein.

It is a general object of the present invention to provide asubstantially purified and isolated nucleic acid sequence which encodesfor the MART-1 melanoma antigen.

It is another object of this invention to provide a recombinant moleculecomprising a vector and all or part of the nucleic acid sequenceencoding MART-1.

It is another object of this invention to produce recombinant proteinsencoded by all or part of the nucleic acid sequence encoding MART-1.

It is a further object of this invention to provide monoclonal orpolyclonal antibodies reactive with the MART-1 protein, peptides orportions thereof.

It is an object of this invention to provide methods of detecting theMART-1 gene or MART-1 mRNA in a biological sample.

It is another object of this invention to provide methods of detectingthe MART-1 protein or peptides in a biological sample.

It is an object of this invention to provide diagnostic methods forhuman disease, in particular for melanomas and metastatic melanomas.

It is a further object of this invention to provide methods forprophylactic or therapeutic uses involving all or part of the nucleicacid sequence encoding MART-1 and its corresponding protein or peptidesderived from the MART-1 amino acid sequence.

It is also an object of this invention to provide melanoma vaccinescomprising all or part of the nucleic acid sequence encoding MART-1 orits corresponding protein for preventing or treating melanoma.

It is a further object of this invention to provide immunogenic peptidesderived from the MART-1 protein sequence for use in vaccines.

It is yet another object of this invention to provide peptides derivedfrom the MART-1 protein sequence which have been modified to increasetheir immunogenicity or enhance induction of antimelanoma immuneresponse by enhancing their binding to MHC molecules, for use in theprophylactic or therapuetic mothods described herein.

In addition, it is another object of this invention to providemultivalent vaccines comprising all or part of the MART-1 nucleic acidsequence or its corresponding protein or peptides and at least one otherimmunogenic molecule capable of eliciting the production of antibodiesin a mammal to melanoma antigens.

It is another object of this invention to provide a method forpreventing or treating melanoma utilizing all or part of the MART-1nucleic acid sequence or its corresponding protein in gene therapyprotocols.

It is a further object of this invention to provide immunogenic peptidesderived from a gp100 melanoma antigen protein sequence for use invaccines.

It is yet another object of this invention to provide peptides derivedfrom a gp100 melanoma antigen sequence which have been modified toincrease their immunogenicity or enhance induction of antimelanomaimmune response by enhancing binding to MHC molecules for use in theprophylactic and therapeutic methods described herein

It is yet another object of this invention to provide a method ofprophylactic or therapeutic immunization for melanoma using the vaccinesdescribed herein.

It is a further object of this invention to provide a method ofidentifying melanoma antigens that would constitute potential targetsfor immunotherapy.

It is yet another object of this invention to provide a method ofidentifying candidate immunogenic peptides derived from either theMART-1 or gp100 sequences for use in immunotherapy.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide and predicted amino acid sequence of thecDNA encoding the MART-1 antigen. The hydrophobic region is underlined.

FIG. 2 shows titration of MART-1 peptides for recognition by TIL. T2cells were incubated with varied concentrations of the purified MART-1peptides, M9-1, M9-2, M9-3, M10-2, M10-3, M10-4 and M10-5, and lysis byTIL clone A42 or TIL line TIL1235 was measured by 4 h-⁵¹Cr releasecytotoxicity assay at an E (EFFECTOR):T (TARGET) ratio of 20:1 for A42and 40:1 for TIL1235. Peptide M9-2 sensitized T2 cells at aconcentration of 1 ng/ml. The purified peptide M10-4 was recognized byTIL1235, but not by A42. (M9-1 |-|, M9-2 -, M9-3, ▪-▪, M10-2 ▴-▴,M10-3 ▾-▾, M10-4 -, M10-5 +-+).

FIG. 3A shows a radionuclide scan of patient 1200 with metastaticmelanoma after receiving the adoptive transfer of autologous ¹¹¹Inlabeled TIL1200. The arrow indicates one of the areas of TILaccumulation corresponding to a metastatic lesion in the left thigh.

FIG. 3B shows regression of subcutaneous metastatic tumors followingtreatment with TIL1200 plus IL-2. Treatment began on day 0.

FIG. 4 show the nucleic acid sequence of the full length cDNA25. Thestart and stop codons are underlined.

FIG. 5A shows amino acid sequence of the full length cDNA25. Theantigenic peptide is underlined.

FIG. 5B shows comparison of the amino acid sequence of the full lengthcDNA25 (cDNA25FL), the truncated form of cDNA25 (cDNA25TR), Pmel17, ME20and gp100. (•indicates deletion; - indicates identity).

FIG. 6 shows northern blot analysis of melanoma and neonatal melanocytecell lines and various fresh tissues (10-20 μg of total RNA) with acDNA25 probe(the Sal I digested fragment of pCRII-cDNA25) and theβ-actin probe(Clontech). C32, 586mel melanoma cell lines and NHEM529,NHEM530 neonatal melanocyte cell lines were very weak positive.

FIGS. 7A-7B show the location of gp100 epitopes and the DNA fragmentstested for epitope analysis and recognition by CTL. FIG. 7A. Five DNAfragments (D3, D5, D4, C4, 25TR) tested for epitope analysis are shown( - - - , identical amino acid). Locations of the identified epitopesare underlined. FIG. 7B. Recognition by CTL (620-1, 620-2, 660-1, 1143,1200) of COS 7 cells transfected with each DNA fragment in pcDNA3plasmid along with HLA-A2.1 cDNA by IFN-γ secretion assays are shown (+,recognized; −, not recognized).

FIGS. 8A-8D show titration of gp100 epitopes by sensitization ofHLA-A2.1+T2 cells for CTL lysis. Lysis of T2 cells preincubated withpeptides was tested in a 4 h ⁵¹Cr release cytotoxicity assay. FIG. 8A,Lysis by TIL1200 of T2 cells incubated with G9₁₅₄(▪) or G₁₀ ₁₅₄(). FIG.8B. Lysis by TIL620 of T2 cells incubated with G9₂₀₉(▪) or G10₂₀₈()FIG. 8C. Lysis by TIL660-1 of T2 cells incubated with G9₂₈₀(▪) FIG. 8D.Lysis by TIL660-2 of T2 cells incubated with G10-5(▪).

DETAILED DESCRIPTION OF THE INVENTION

For the purpose of a more complete understanding of the invention, thefollowing definitions are described herein. Nucleic acid sequencesincludes, but is not limited to, DNA, RNA or cDNA. Nucleic acid sequenceas used herein refers to an isolated and purified nucleic acid sequence.MART-1 messenger RNA (mRNA) refers to one or more RNA transcripts whichare a product of the MART-1 gene. Substantially homologous as usedherein refers to substantial correspondence between the nucleic acidsequence of MART-1 shown in FIG. 1 (SEQ ID NO:1) and that of any othernucleic acid sequence. Substantially homologous means about 50-100%homologous homology, preferably by about 70-100% homology, and mostpreferably about 90-100% homology between the MART-1 sequence and thatof any other nucleic acid sequence. In addition, substantiallyhomologous as used herein also refers to substantial correspondencesbetween the amino acid sequence of the MART-1 antigen shown in FIG. 1(SEQ ID NO:2) and that of any other amino acid sequence.

Major Histocompatibility Complex (MHC) is a generic designation meant toencompass the histo-compatibility antigen systems described in differentspecies including the human leucocyte antigens (HLA).

The term melanoma includes, but is not limited to, melanomas, metastaticmelanomas, melanomas derived from either melanocytes or melanocytesrelated nevus cells, melanocarcinomas, melanoepitheliomas,melanosarcomas, melanoma in situ, superficial spreading melanoma,nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma,invasive melanoma or familial atypical mole and melanoma (FAM-M)syndrome. Such melanomas in mammals may be caused by, chromosomalabnormalities, degenerative growth and developmental disorders,mitogenic agents, ultraviolet radiation (UV), viral infections,inappropriate tissue expression of a gene, alterations in expression ofa gene, or carcinogenic agents. The aforementioned melanomas can bediagnosed, assessed or treated by methods described in the presentapplication.

By atypical mole we mean a mole with features that are abnormal and maybe precancerous.

By melanoma antigen or immunogen we mean all or parts thereof of theMART-1 protein or peptides based on the MART-1 protein sequence capableof causing a cellular or humoral immune response in a mammal. Suchantigens may also be reactive with antibodies from animals immunizedwith all, part or parts of the MART-1 protein (SEQ ID NO:2). Such aprotein or peptide may be encoded by all or part of the MART-1 nucleicacid sequence of this invention.

By immunogenic peptide we mean a peptide derived from the MART-1 proteinsequence (FIG. 1; SEQ ID NO. 2) or a gp100 protein sequence (FIG. 5A;SEQ ID NO: 27) capable of causing a cellular or humoral immune responsein a mammal. Such peptides may be reactive with antibodies from ananimal immunized with the peptides. Such peptides are about 5-20 aminoacid in length preferably about 8 to 15 amino acids in length, and mostpreferably about 9-10 amino acids in length.

One skilled in the art will understand that the bioassays of the presentinvention may be used in the analysis of biological samples or tissuesfrom any vertebrate species. In a preferred embodiment, mammalianbiological samples or tissues are analyzed.

Tissue includes, but is not limited to, single cells, whole organs andportions thereof. Biological samples include, but are not limited to,tissues, primary cultures of mammalian tissues, biopsy specimens,pathology specimens, and necropsy specimens. Mammal includes but is notlimited to, humans, monkeys, dogs, cats, mice, rats, pigs, cows, pigs,horses, sheep and goats.

The present invention provides a nucleic acid sequence which encodes anovel melanoma antigen recognized by T cells. This novel melanomaantigen designated MART-1 (melanoma antigen-recognized by T-Cells-1).MART-1 shows no significant homology to any known melanoma antigen andthus represents a new melanoma antigen. The MART-1 antigen contains ahighly hydrophobic region from amino acids 27 to 47 (SEQ ID. NO:2)followed by three arginine residues, suggestive of a transmembraneprotein. Although no significant homology exists to the entire proteinthere is a 27 amino acid segment (amino acids 57-83; SEQ ID. NO:2) thatis 37% identical to a Type II membrane protein previously recognized asmouse natural killer cell surface protein NKR-P1 (Yokoyama, W. M., etal. (1991), J. Immunol. 147:3229-3236). MART-1 does not contain a leadersequence characteristic of many Type I membrane proteins (Singer, S. J.(1990) Annu. Rev. Cell Biol. 6: 247-296).

MART-1 RNA expression appears to be restricted to fresh and culturedmelanoma and melanocyte cell lines and human retina; expression has notbeen found in any other fresh or cultured tissues or other tumorhistologies tested. The cDNA sequence for MART-1 is shown in FIG. 1 (SEQID NO:1), the deduced amino acid sequence for the MART-1 protein is alsoshown in FIG. 1 (SEQ ID NO.:1).

The nucleic acid sequence for MART-1 shown in FIG. 1 (SEQ ID NO.:1),represents a preferred embodiment of the invention. It is, however,understood by one skilled in the art that due to the degeneracy of thegenetic code variations in the cDNA sequence shown in FIG. 1 (SEQ IDNO.:1) will still result in a DNA sequence capable of encoding theMART-1 protein antigen. Such DNA sequences are therefore functionallyequivalent to the sequence set forth in FIG. 1 (SEQ ID NO.:1) and areintended to be encompassed within the present invention. Further, aperson of skill in the art will understand that there are naturallyoccurring allelic variations in a given species of the MART-1 nucleicacid sequence shown in FIG. 1 (SEQ ID NO.:1), these variations are alsointended to be encompassed by the present invention.

The predicted MART-1 antigen is a 118 amino acid protein of about 13(kd). This invention further includes MART-1 protein or peptides oranalogs thereof having substantially the same function as the MART-1antigen or protein of this invention. Such proteins or polypeptidesinclude, but are not limited to, a fragment of the protein, or asubstitution, addition or deletion mutant of the MART-1 protein. Thisinvention also encompasses proteins or peptides that are substantiallyhomologous to the MART-1 antigen. Substantially homologous means about50-100% homology, preferably about 70-100% homology, and most preferablyabout 90-100% homology between the MART-1 and any another amino acidsequence or protein or peptide.

The term “analog” includes any polypeptide having an amino acid residuesequence substantially identical to the MART-1 sequence specificallyshown herein (FIG. 1; SEQ ID NO: 1) in which one or more residues havebeen conservatively substituted with a functionally similar residue andwhich displays the functional aspects of the MART-1 antigen as describedherein. Examples of conservative substitutions include the substitutionof one non-polar (hydrophobic) residue such as isoleucine, valine,leucine or methionine for another, the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, between glycine and serine, thesubstitution of one basic residue such as lysine, arginine or histidinefor another, or the substitution of one acidic residue, such as asparticacid or glutamic acid or another.

The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residue.“Chemical derivative” refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Examples of such derivatized molecules include for example, thosemolecules in which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as chemical derivatives are those proteins or peptideswhich contain one or more naturally-occurring amino acid derivatives ofthe twenty standard amino acids. For examples: 4-hydroxyproline may besubstituted for proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.Proteins or polypeptides of the present invention also include anypolypeptide having one or more additions and/or deletions or residuesrelative to the sequence of a polypeptide whose sequence is encoded isthe DNA of MART-1, so long as the requisite activity is maintained.

This invention also provides a recombinant DNA molecule comprising allor part of the MART-1 nucleic acid sequence (SEQ. ID NO.:1) and avector. Expression vectors suitable for use in the present invention maycomprise at least one expression control element operationally linked tothe nucleic acid sequence. The expression control elements are insertedin the vector to control and regulate the expression of the nucleic acidsequence. Examples of expression control elements include, but are notlimited to, lac system, operator and promoter regions of phage lambda,yeast promoters and promoters derived from polyoma, adenovirus,retrovirus or SV40. Additional preferred or required operationalelements include, but are not limited to, leader sequence, terminationcodons, polyadenylation signals and any other sequences necessary orpreferred for the appropriate transcription and subsequent translationof the nucleic acid sequence in the host system. It will be understoodby one skilled in the art the correct combination of required orpreferred expression control elements will depend on the host systemchosen. It will further be understood that the expression vector shouldcontain additional elements necessary for the transfer and subsequentreplication of the expression vector containing the nucleic acidsequence in the host system. Examples of such elements include, but arenot limited to, origins of replication and selectable markers. It willfurther be understood by one skilled in the art that such vectors areeasily constructed using conventional methods (Ausubel et al., (1987) in“Current Protocols in Molecular Biology”, John Wiley and Sons, New York,N.Y.) or commercially available.

Another aspect of this invention relates to a host organism into whichrecombinant expression vector containing all or part of the MART-1nucleic acid sequence has been inserted. The host cells transformed withthe MART-1 nucleic acid sequence of this invention include eukaryotes,such as animal, plant, insect and yeast cells and prokaryotes, such asE. coli. The means by which the vector carrying the gene may beintroduced into the cell include, but are not limited to,microinjection, electroporation, transduction, or transfection usingDEAE-dextran, lipofection, calcium phosphate or other procedures knownto one skilled in the art (Sambrook et al. (1989) in “Molecular Cloning.A Laboratory Manual”, Cold Spring Harbor Press, Plainview, N.Y.).

In a preferred embodiment, eukaryotic expression vectors that functionin eukaryotic cells are used. Examples of such vectors include, but arenot limited to, retroviral vectors, vaccinia virus vectors, adenovirusvectors, herpes virus vector, fowl pox virus vector, bacterialexpression vectors, plasmids, such as pcDNA3 (Invitrogen, San Diego,Calif.) or the baculovirus transfer vectors. Preferred eukaryotic celllines include, but are not limited to, COS cells, CHO cells, HeLa cells,NIH/3T3 cells, 293 cells (ATCC#CRL1573), T2 cells, dendritic cells, ormonocytes. In a particularly preferred embodiment the recombinant MART-1protein expression vector is introduced into mammalian cells, such asNIH/3T3, COS, CHO, 293 cells (ATCC #CRL 1573), T2 cells, dendriticcells, or monocytes to ensure proper processing and modification of theMART-1 protein. In an alternative embodiment the MART-1 cDNA isintroduced into COS7 (Gluzman, Y. et al. (1981) Cell 23: 175-182). Thechoice of an appropriate cell is within the skill of a person in theart.

In one embodiment the expressed recombinant MART-1 protein may bedetected by methods known in the art which include Coomassie bluestaining and Western blotting using antibodies specific for the MART-1protein.

In a further embodiment, the recombinant protein expressed by the hostcells can be obtained as a crude lysate or can be purified by standardprotein purification procedures known in the art which may includedifferential precipitation, molecular sieve chromatography, ion-exchangechromatography, isoelectric focusing, gel electrophoresis, affinity, andimmunoaffinity chromatography and the like. (Ausubel et. al., (1987) in“Current Protocols in Molecular Biology” John Wiley and Sons, New York,N.Y.). In the case of immunoaffinity chromatography, the recombinantprotein may be purified by passage through a column containing a resinwhich has bound thereto antibodies specific for the MART-1 protein(Ausubel et. al., (1987) in “Current Protocols in Molecular Biology”John Wiley and Sons, New York, N.Y.).

The nucleic acid sequence or portions thereof, of this invention areuseful as probes for the detection of expression of the MART-1 gene innormal and diseased tissue. Therefore, another aspect of the presentinvention relates to a bioassay for detecting messenger RNA encoding theMART-1 protein in a biological sample comprising the steps of (a)contacting all or part of the nucleic acid sequence of this inventionwith said biological sample under conditions allowing a complex to formbetween said nucleic acid sequence and said messenger RNA, (b) detectingsaid complexes and, (c) determining the level of said messenger RNA.

RNA can be isolated as whole cell RNA or as poly(A)⁺ RNA. Whole cell RNAcan be isolated by a variety of methods known to those skilled in theart. (Ausubel et al., (1987) on “Current Protocols in MolecularBiology”, John Wiley and Sons, New York). Such methods includeextraction of RNA by differential precipitation (Birnboim, H. C. (1988)Nucleic Acids Res., 16:1487-1497), extraction of RNA by organic solvents(Chomczynski, P. et al. (1987) Anal. Biochem., 162:156-159) and theextraction of RNA with strong denaturants (Chirgwin, J. M. et al. (1979)Biochemistry, 18:5294-5299). Poly(A)⁺ RNA can be selected from wholecell RNA by affinity chromatography on oligo-d(T) columns (Aviv, H. etal. (1972) Proc. Natl. Acad. Sci., 69:1408-1412). Examples of methodsfor determining cellular messenger mRNA levels for step (c) include, butare not limited to Northern blotting (Alwine, J. C. et al. (1977) Proc.Natl. Acad. Sci., 74:5350-5354), dot and slot hybridization (Kafatos, F.C. et al. (1979) Nucleic Acids Res., 7:1541-1522), filter hybridization(Hollander, M. C. et al. (1990) Biotechniques; 9:174-179), RNaseprotection (Sambrook et. al., (1989) in “Molecular Cloning, A LaboratoryManual”, Cold Spring Harbor Press, Plainview, N.Y.), polymerase chainreaction (Watson, J. D. et al. (1992) in “Recombinant DNA” SecondEdition, W. H. Freeman and Company, New York) and nuclear run-off assays(Ausubel et. al., (1987) in “Current Protocols in Molecular Biology”Supplement 9 (1990); John Wiley and Sons, New York, N.Y.).

Detection of complexes in Step (b) of the bioassay can also be carriedout by a variety of techniques. Detection of the complexes by signalamplification can be achieved by several conventional labellingtechniques including radiolabels and enzymes (Sambrook et. al., (1983)in “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Press,Plainview, N.Y.; Ausubel et al., (1987) in “Current Protocols inMolecular Biology, John Wiley and Sons, New York N.Y.). Radiolabellingkits are also commercially available. The MART-1 nucleic acid sequenceused as a probe in step(c) of the bioassay may be RNA or DNA. Preferredmethods of labelling the DNA sequences are with ³²P using Klenow enzymeor polynucleotide kinase. Preferred methods of labeling RNA or riboprobesequences are with ³²P or ³⁵S using RNA polymerases. In addition, thereare known non-radioactive techniques for signal amplification includingmethods for attaching chemical moieties to pyrimidine and purine rings(Dale, R. N. K. et al. (1973) Proc. Natl. Acad. Sci., 70:2238-2242;Heck, R. F. (1968) S. Am. Chem. Soc., 90:5518-5523), methods which allowdetection by chemiluminescence (Barton, S. K. et al. (1992) J. Am. Chem.Soc., 114:8736-8740) and methods utilizing biotinylated nucleic acidprobes (Johnson, T. K. et al. (1983) Anal. Biochem., 133:125-131;Erickson, P. F. et al. (1982) J. of Immunology Methods, 51:241-249;Matthaei, F. S. et al (1986) Anal. Biochem., 157:123-128) and methodswhich allow detection by fluorescence using commercially availableproducts. Non-radioactive labelling kits are also commerciallyavailable.

Examples of biological samples that can be used in this bioassayinclude, but are not limited to, primary mammalian cultures, continuousmammalian cell lines, such as melanocyte cell lines, mammalian organssuch as skin or retina, tissues, biopsy specimens, neoplasms, pathologyspecimens, and necropsy specimens.

In a preferred embodiment, a ³²P radiolabelled MART-1 probe, asexemplified in Example 1, is used. Preferably the MART-1 probe is thefull length cDNA in FIG. 1 (SEQ ID NO:1). The approximately 1.6 Kilobase(kb) cDNA (FIG. 1; SEQ ID NO:1) was cloned into the vector and theresulting plasmid deposited with the American Type Culture Collection(ATCC) 12301 Parklawn Drive, Rockville, Md. 20852 USA on Apr. 14, 1994,and given ATCC Deposit Number 75738. The full length MART-1 nucleic acidsequence can be isolated from the pCRII plasmid by digestion withHINDIII and XhoI restriction enzymes. This 1.6 kb nucleic acid sequencecan then be used as a probe. This probe is used to detect MART-1 mRNA intotal RNA or poly A⁺ RNA isolated from a variety of tissues orbiological samples.

In another embodiment, combinations of oligonucleotide pairs based onthe MART-1 sequence in FIG. 1 (SEQ ID NO.:1) are used as PolymeraseChain Reaction (PCR) primers to detect MART-1 mRNA in a biologicalsample. These primers can be used in a method following the reversetranscriptase-Polymerase Chain Reaction (RT-PCR) process for amplifyingselected RNA nucleic acid sequences as detailed in Ausubel et al., (eds)(1987) In “Current Protocols in Molecular Biology” Chapter 15, JohnWiley and Sons, New York, N.Y. The oligonucleotides can be synthesizedby automated instruments sold by a variety of manufacturers or can becommercially prepared based upon the nucleic acid sequence of thisinvention. One skilled in the art will know how to select PCR primersbased on the MART-1 nucleic acid sequence for amplifying MART-1 RNA in asample.

The MART-1 nucleic acid sequence or portions thereof (FIG. 1: SEQ IDNO:1) of this invention are useful to detect alterations of the MART-1gene in normal or diseased mammalian tissue. By alteration, we meanadditions, deletions, substitutions or duplications in the MART-1 genesequence or gene amplification of the MART-1 gene sequence. Therefore,another aspect of the present invention relates to an assay fordetecting alterations of the MART-1 gene in a biological samplecomprising the steps of (a) contacting all or part of the nucleic acidsequence of this invention with genomic DNA isolated from a biologicalsample under conditions allowing a complex to form between said nucleicacid sequence and said genomic DNA, (b) detecting said complexes, and(c) determining alterations in said MART-1 gene by comparison to acontrol sample.

Standard methods for isolating DNA from a biological sample, detectingalterations in a gene and detecting complex between the MART-1 nucleicacid probe and genomic DNA sequences are provided in manuals such asSambrook et al., (eds) (1989) “Molecular Cloning, A Laboratory Mineral”,Cold Spring Harbor Press, Plainview, N.Y. and in Ausubel et al., (eds)(1987) in “Current Protocols in Molecular Biology” John Wiley and Sons,New York, N.Y.

The MART-1 nucleic acid sequences of this invention (FIG. 1; SEQ IDNo:1) can also be used as probes to isolate the MART-1 homologs in otherspecies. In a preferred embodiment the MART-1 cDNA (FIG. 1; SEQ ID No:1)is used to screen a mammalian cDNA library, positive clones are selectedand sequenced. Examples of tissue sources from which the cDNA librarycan be synthesized include, but are not limited to skin, retina,melanocytes, neonatal skin and embryos. Preferably a melanoma library isscreened using the MART-1 cDNA as a probe (FIG. 1; SEQ ID No. 1). Oneskilled in the art will understand the appropriate hybridizationconditions to be used to detect the homologs. Conventional methods fornucleic acid hybridization, construction of libraries and cloningtechniques are described in Sambrook et al., (eds) (1989) In “MolecularCloning A Laboratory Manual” Cold Spring Harbor Press, Plainview, N.Y.and Ausubel et al., (eds) in “Current Protocols in Molecular Biology”(1987), John Wiley and Sons, New York, N.Y.

It is known that all or parts thereof of the MART-1 protein is anantigen present on melanoma cells. It is therefore another aspect ofthis invention to provide MART-1 nucleic acid probes to be utilized indetecting MART-1 RNA or alterations in the level of MART-1 mRNA inbiological sample isolated from a mammal afflicted with a disease.Examples of such diseases, include but are not limited to, melanomas. Byalterations in the level of MART-1 mRNA we mean an increase or decreasein the level of an RNA relative to a control sample or the appearance ordisappearance of the MART-1 mRNA relative to a control sample. Detectionin the alterations of MART-1 mRNA will allow for diagnosis or theassessment of the diseased state. Therefore, alterations in the level ofMART-1 mRNA may be predictive of the prognosis for the afflicted mammal.

In another embodiment the nucleic acid of this invention can be used inin situ hybridization on mammalian tissues to determine the precise siteor subcellular site of expression of the MART-1 gene within a tissue. Apreferred method of labeling the MART-1 nucleic acid sequence issynthesizing a ³⁵S-labeled RNA probe by in vitro transcription utilizingSP6 polymerase. In the MART-1 plasmid (ATCC Deposit #75738) the sensestrand is under the control of the T7 promoter, the antisense strand isunder the SP6 promoter. It is preferable that the probe be hydrolyzed toa probe length of approximately 400-200 base pairs. Conventional methodsfor preparation of tissues for in situ, synthesis of probes anddetection of signal can be found in Ausubel et. al., (eds) (1987) in“Current Protocols in Molecular Biology,” John Wiley and Sons, New York,N.Y. Chapter 14 and Vander Ploeg, M., Raap A. K. (1988) In “NewFrontiers in Cytology” Goerttler, K., Feichter, G E, Witte. S. (eds) pp13-21 Springer-Verlag, New York. The probe is then contacted withmammalian tissue sections and in situ analyses performed by conventionalmethods. Examples of tissues that can be used include, but are notlimited to, mammalian embryos, adult mammalian tissues, such as skin,lymph nodes and retina, biopsy specimens, pathology specimens andnecropsy specimens. In a preferred embodiment, MART-1 in situ probes maybe used to evaluate MART-1 RNA expression in diseased tissue forinvasive early melanoma to characterize radial and vertical growthphases of the melanoma lesion and assess the margins of the diseasewithin the tissue.

In yet another embodiment of this invention all or parts thereof of theMART-1 (SEQ ID NO:1) nucleic acid sequence can be used to generatetransgenic animals. Preferably the MART-1 gene is introduced into ananimal or an ancestor of the animal at an embryonic stage, preferably atthe one cell stage and generally not later than about the eight cellstage. There are several means by which transgenic animals carrying aMART-1 gene can be made. One method involves the use of retrovirusescarrying all or part of the MART-1 sequence. The retroviruses containingthe transgene are introduced into the embryonic animal by transfection.Another methods involves directly injecting the transgene into theembryo. Yet another methods employs the embryonic stem cell method orhomologous recombination method known to workers in the field. Examplesof animals into which the MART-1 transgene can be introduced include butare not limited to, primates, mice, rats or other rodents. Suchtransgenic animals may be useful as biological models for the study ofmelanoma and to evaluate diagnostic or therapeutic methods for melanoma.

This invention further comprises an antibody or antibodies reactive withthe MART-1 protein or peptides or modified peptides or analogs thereofhaving the amino acid sequence defined in FIG. 1 (SEQ ID NO: 2) or aunique portion thereof. In this embodiment of the invention theantibodies are monoclonal or polyclonal in origin. MART-1 protein orpeptides used to generate the antibodies may be from natural orrecombinant sources or generated by chemical synthesis. Natural MART-1proteins can be isolated from mammalian biological samples. Biologicalsamples include, but are not limited to mammalian tissues such as freshmelanoma, skin, retina, primary or continuous cultures of mammaliancells such as melanoma cultures or cultured melanocytes. The naturalMART-1 proteins may be isolated by the same methods described above forrecombinant proteins. Recombinant MART-1 proteins or peptides may beproduced and purified by conventional methods. Synthetic MART-1 peptidesmay be custom ordered or commercially made based on the predicted aminoacid sequence of the present invention (FIG. 1; SEQ ID:2) or synthesizedby methods known to one skilled in the art (Merrifield, R. B. (1963) J.Amer. Soc. 85:2149). Examples of MART-1 peptides include, but are notlimited to, are AAGIGILTV (M9-2; SEQ. ID NO. 4), EAAGIGILTV (M10-3; SEQ.ID NO. 17) and AAGIGILTVI (M10-4; SEQ. ID NO. 18) (peptides arepresented in single letter amino acid code). The most preferred peptideis AAGIGILTV (SEQ ID NO:4).

Alternatively, peptides derived form the MART-1 protein sequence maymodified to increase their immunogenicity by enhancing binding of thepeptide to the MHC molecules in which the peptide is presented. Examplesof such modified MART-1 peptides that may used are shown, but notlimited to, the peptides in Table 14. In a preferred embodiment theMART-1 peptide that is modified to enhance its binding to MHC Class Imolecules is AAGIGILTV (SEQ ID NO:4). By way of example, the modifiedpeptides ALGIGILTV (M9-2-2L), WAGIGILTV (M9-2-1W), FAGIGILTV (M9-2-1F)and AAYIGILTV (M9-2-3Y). The peptide or modified peptide may beconjugated to a carrier molecule to enhance the antigenicity of thepeptide. Examples of carrier molecules, include, but are not limited to,human albumin, bovine albumin, lipoprotein and keyhole limpethemo-cyanin (“Basic and Clinical Immunology” (1991) Stites, D. P. andTerr A. I. (eds) Appleton and Lange, Norwalk Connecticut, San Mateo,Calif.).

Exemplary antibody molecules for use in the detection methods of thepresent invention are intact immunoglobulin molecules, substantiallyintact immunoglobulin molecules or those portions of an immunoglobulinmolecule that contain the antigen binding site, including those portionsof immunoglobulin molecules known in the art as F(ab), F(ab′); F(ab)₂and F(v). Polyclonal or monoclonal antibodies may be produced by methodsknown in the art. (Kohler and Milstein (1975) Nature 256, 495-497;Campbell “Monoclonal Antibody Technology, the Production andCharacterization of Rodent and Human Hybridomas” in Burdon et al. (eds.)(1985) “Laboratory Techniques in Biochemistry and Molecular Biology,”Volume 13, Elsevier Science Publishers, Amsterdam). The antibodies orantigen binding fragments may also be produced by genetic engineering.The technology for expression of both heavy and light chain genes in E.coli is the subject of the PCT patent applications: publication numberWO 901443, WO 901443 and WO 9014424 and in Huse et al. (1989) Science246:1275-1281.

The antibodies of this invention may react with native or denaturedMART-1 protein, peptides or analogs thereof, or modified peptides ananalogs thereof. The specific immunoassay in which the antibodies are tobe used will dictate which antibodies are desirable. Antibodies may beraised against the MART-1 protein or portions thereof or againstsynthetic peptides homologous to the MART-1 amino acid sequence.

In one embodiment the antibodies of this invention are used inimmunoassays to detect the novel MART-1 protein in biological samples.In this method the antibodies of the present invention are contactedwith a biological sample and the formation of a complex between theMART-1 antigen and antibody is detected. Immunoassays of the presentinvention may be radioimmunoassay, Western blot assay, immunofluorescentassay, enzyme immunoassay, chemiluminescent assay, immunohistochemicalassay and the like. (In “Principles and Practice of Immunoassay” (1991)Christopher P. Price and David J. Neoman (eds), Stockton Press, NewYork, N.Y.; Ausubel et al. (eds) (1987) in “Current Protocols inMolecular Biology” John Wiley and Sons, New York, N.Y.). Standardtechniques known in the art for ELISA are described in Methods inImmunodiagnosis, 2nd Edition, Rose and Bigazzi, eds., John Wiley andSons, New York 1980 and Campbell et al., Methods of Immunology, W. A.Benjamin, Inc., 1964, both of which are incorporated herein byreference. Such assays may be direct, indirect, competitive, ornoncompetitive immunoassays as described in the art (In “Principles andPractice of Immunoassay” (1991) Christopher P. Price and David J. Neoman(eds), Stockton Pres, NY, N.Y.; Oellirich, M. 1984. J. Clin. Chem. Clin.Biochem. 22: 895-904) Biological samples appropriate for such detectionassays include mammalian tissues, melanoma and melanocyte cell lines,skin, retina, lymph nodes, pathology specimens, necropsy specimens, andbiopsy specimens. Proteins may be isolated from biological samples byconventional methods described in (Ausubel et al., (eds) (1987) in“Current Protocols in Molecular Biology” John Wiley and Sons, New York,N.Y.).

The antibodies of this invention can therefore be used in immunoassaysto detect MART-1 antigen or alteration in the level of expression of theMART-1 antigen in biological samples isolated from mammals afflictedwith a disease or disorder. Examples of biological samples include, butare not limited to, mammalian tissues, biopsy tissue samples, melanomaand lymph node biopsy samples, pathology and tissue samples. Examples ofdiseases that can be assessed by these immunoassays, include, but arenot limited to, melanomas and tissues which are secondary sites formelanoma metastasis. By alteration in level of expression, we mean anincrease or decrease of the MART protein or portions thereof relative toa control sample. Alteration is also meant to encompass substitution,deletion or addition mutants of the MART-1 protein. Such mutations canbe determined by using the antibodies of this invention known to reactwith specific epitopes of the MART-1 protein and determining whichepitopes are present relative to a control. The antibodies of thisinvention can therefore be used in an immunoassay to diagnose, assess orprognoses a mammal afflicted with the disease.

In a preferred embodiment, the MART-1 antibodies of this invention areused to assess the presence of the MART-1 antigen from a tissue biopsyof a mammal afflicted with melanoma using immunocytochemistry. Suchassessment of the delineation of the MART-1 antigen in a diseased tissuecan be used to prognose the progression of the disease in a mammalafflicted with the disease. Specifically the MART-1 antibodies can beused to characterize the radial and vertical growth phases of themelanoma lesion. Conventional methods for immunohistochemistry aredescribed in (Harlow and Lane (eds) (1988) In “Antibodies A LaboratoryManual”, Cold Spinning Harbor Press, Cold Spring Harbor, N.Y.; Ausbel etal. (eds) (1987). In Current Protocols In Molecular Biology John Wileyand Sons (New York, N.Y.).

In another embodiment, antibodies of this invention may be used topurify the MART-1 protein or portions thereof. Immunoaffinitychromatography can be performed by conventional methods known to oneskilled in the art (Ausubel et al. (eds) (1987) in “Current Protocols inMolecular Biology” John Wiley and Sons, New York, N.Y.).

In another preferred embodiment rabbit antisera containing antibodieswhich specifically recognize the MART-1 protein is used to detect saidprotein in Western Blot Analysis. Such antisera is directed to all, or apart or parts of the MART-1 protein or synthetic peptides derived fromthe MART-1 protein sequence. Preferably a MART-1 synthetic peptidederived from the MART-1 predicted amino acid sequence is used (FIG. 1;SEQ ID NO:2). Alternatively, modified MART-1 peptides may be used. Thepeptide is synthesized by standard methods on an automated peptidesynthesizer and purified by high pressure liquid chromatography (HPLC)as described in Example 2. The purified peptide may be conjugated to acarrier as described in (M. Bodanszky (1984) “Principles of PeptideSynthesis,” Springer Verlag, New York, N.Y.). Using conventionalmethods, rabbits may be immunized with the MART-1 protein or peptideconjugated to carriers. Preferably about 0.1 to about 10 (mg) of antigenin adjuvant may be used, most preferably about 1 mg of antigen inadjuvant may be used. The animal receives similar booster doses andantisera titer is assessed by ELISA assay. Satisfactory levels ofantisera are obtained when the anti-peptide antibody titer reaches aplateau. This antibody can be used in the standard immunoassaysdescribed above.

T-lymphocytes recognize antigen in association with Class I or Class IIMHC molecules in the form of a peptide fragment bound to an MHCmolecule. The degree of peptide binding to a given MHC allele is basedon amino acids at particular positions within the peptide (Parker et al.(1992) Journal of Immunology 149:3580; Kubo, et al. (1994) Journal ofImmunology 52:3913-3924; Ruppert J. et al. (1993) Cell 74:929-937; Falket al. (1991) Nature 351:290-296, each of which is herein incorporatedby reference). Therefore, another embodiment of this invention relatesto peptides derived from the MART-1 protein sequence (FIG. 1; SEQ IDNO:2) which have been modified to increase immunogenicity by enhancingbinding of the peptide to the MHC molecule with which the peptide isassociated. By way of example, modification may include substitution,deletion or addition of an amino acid in the given-immunogenic peptidesequence or mutation of existing amino acids within the givenimmunogenic peptide sequence, or derivatization of existing amino acidswithin the given immunogenic peptide sequence. Any amino acid comprisingthe immunogenic peptide sequence may be modified in accordance with thisinvention. In a preferred embodiment at least one amino acid issubstituted or replaced within the given immunogenic peptide sequence.Any amino acid may be used to substitute or replace a given amino acidwithin the immunogenic peptide sequence. Modified peptides are intendedto include any immunogenic MART-1 peptide which has been modified andexhibits enhanced binding to the MHC molecule with which it associateswhen presented to the T-cell.

By way of example, the HLA-A2 allele binds peptides of nine or ten aminoacids. Examples of positions within the peptide that may be altered toenhance binding include, but are not limited to, the first position, thesecond position, the third position and the last position of thepeptide. Any amino acid may be used to substitute or replace thesepositions within the immunogenic peptide sequence. For enhanced bindingto HLA-A2 the amino acid at the second position of the peptide ispreferably a hydrophobia aliphatic amino acid. Examples or amino acidsthat may be used at the second position include, but are not limited to,leucine, methionine, alanine, isoleucine, valine, threonine or glycine.Preferably leucine or methionine is found at the second position of thepeptide. The last amino acid of the peptide (either the 9th or 10thamino acid depending on the length of the peptide) is preferably ahydrophobic aliphatic amino acid. Examples of amino acids that may beused in the last position of the peptide include, but are not limitedto, valine, methionine, leucine, alanine, isoleucine, threonine orglycine. Preferably valine is found at the last position in the peptide.The amino acids at the first and third positions in the peptide may alsobe modified to enhance binding of the peptide to the MHC Class Imolecule. The amino acids at the first and third positions in thepeptide may be any amino acid. Preferably, the amino acids at the firstand third positions are hydrophobic aliphatic amino acids or aromaticamino acids. Examples of amino acids that maybe used at these positionsinclude, but are not limited to, leucine, methionine, valine, alanine,isoleucine, threonine, glycine, tryptophan, phenylalanine, tyrosine,serine, aspartic acid, or lysine. Examples of MART-1 peptides that maybe modified include, but are not limited to, AAGIGILTV (SEQ ID NO: 4),EAAGIGILTV (SEQ ID NO: 17) and AAGIGILTVI (SEQ ID NO: 18) (peptides arepresented in single letter amino acid code). By way of example theimmunogenic MART-1 peptide AAGIGILTV (SEQ ID NO:4) may be modifiedaccording to the following formula X₁X₂X₃IGILTX₄ wherein

X₁ may be any amino acid, preferably any hydrophobic aliphatic aminoacid, or aromatic amino acid. Examples of amino acids that may be used,but are not limited to, alanine, tryptophan, phenylalanine, tyrosine,lysine, isoleucine, leucine, methionine, threonine, glycine or serine.

X₂ may be any hydrophobic amino acid, preferably an aliphatichydrophobic amino acids. Examples of amino acids that may be usedinclude, but are not limited to, leucine, methionine, isoleucine,valine, threonine, alanine or glycine.

X₃ may be any amino acid, preferably any hydrophobic aliphatic aminoacid, or aromatic amino acid. Examples of amino acids that may be usedinclude, but are not limited to, tryptophan, phenylalanine, tyrosine,lysine, aspartic acid, serine, alanine, glycine, isoleucine, valine, orthreonine.

X₄ may be any hydropbic amino acid, preferably a hydrophobic aliphaticamino acid. Examples of amino acids that may be used include, but arenot limited to, valine, leucine, isoleucine, alanine, threonine, orglycine.

Examples of modified AAGIGILTV (SEQ ID NO:4) peptide sequences that maybe produced are shown but not limited to the peptides in Table 14(Example 5).

This invention further includes analogs of these immunogenic modifiedpeptides derived from the MART-1 amino acid sequence (FIG. 1; SEQ IDNO:2) which have been modified. The term analog is intended to includeany peptide which displays the functional aspects of these modifiedpeptides. The term analog also includes conservative substitutions orchemical derivatives of these modified peptides as described above.These modified peptides may be synthetically or recombinantly producedby conventional methods.

The recombinant or natural MART-1 protein, peptides, or analogs thereof,or modified MART-1 peptides, or analogs thereof may be used as a vaccineeither prophylactically or therapeutically. When providedprophylactically the vaccine is provided in advance of any evidence ofmelanoma. The prophylactic administration of the MART-1 vaccine shouldserve to prevent or attenuate melanoma in a mammal. In a preferredembodiment mammals, preferably human, at high risk for melanoma areprophylactically treated with the vaccines of this invention. Examplesof such mammals include, but are not limited to, humans with a familyhistory of melanoma, humans with a history of atypical moles, humanswith a history of FAM-M syndrome or humans afflicted with melanomapreviously resected and therefore at risk for reoccurrence. Whenprovided therapeutically, the vaccine is provided to enhance thepatient's own immune response to the tumor antigen present on themelanoma or metastatic melanoma. The vaccine, which acts as animmunogen, may be a cell, cell lysate from cells transfected with arecombinant expression vector, cell lysates from cells transfected witha MART-1 recombinant expression vector, or a culture supernatantcontaining the expressed protein. Alternatively, the immunogen is apartially or substantially purified recombinant MART-1 protein, peptideor analog thereof or modified peptides or analogs thereof. The proteinsor peptides may be conjugated with lipoprotein or administered inliposomal form or with adjuvant.

While it is possible for the immunogen to be administered in a pure orsubstantially pure form, it is preferable to present it as apharmaceutical composition, formulation or preparation.

The formulations of the present invention, both for veterinary and forhuman use, comprise an immunogen as described above, together with oneor more pharmaceutically acceptable carriers and, optionally, othertherapeutic ingredients. The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethod well-known in the pharmaceutical art.

All methods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired formulation.

Formulations suitable for intravenous intramuscular, subcutaneous, orintraperitoneal administration conveniently comprise sterile aqueoussolutions of the active ingredient with solutions which are preferablyisotonic with the blood of the recipient. Such formulations may beconveniently prepared by dissolving solid active ingredient in watercontaining physiologically compatible substances such as sodium chloride(e.g. 0.1-2.0M), glycine, and the like, and having a buffered pHcompatible with physiological conditions to produce an aqueous solution,and rendering said solution sterile. These may be present in unit ormulti-dose containers, for example, sealed ampoules or vials.

The formulations of the present invention may incorporate a stabilizer.Illustrative stabilizers are polyethylene glycol, proteins, saccharides,amino acids, inorganic acids, and organic acids which may be used eitheron their own or as admixtures. These stabilizers are preferablyincorporated in an amount of 0.11-10,000 parts by weight per part byweight of immunogen. If two or more stabilizers are to be used, theirtotal amount is preferably within the range specified above. Thesestabilizers are used in aqueous solutions at the appropriateconcentration and pH. The specific osmotic pressure of such aqueoussolutions is generally in the range of 0.1-3.0 osmoles, preferably inthe range of 0.8-1.2. The pH of the aqueous solution is adjusted to bewithin the range of 5.0-9.0, preferably within the range of 6-8. Informulating the immunogen of the present invention, anti-adsorptionagent may be used.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achievedthrough the use of polymer to complex or absorb the proteins or theirderivatives. The controlled delivery may be exercised by selectingappropriate macromolecules (for example polyester, polyamino acids,polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled-release preparations is to incorporate the MART-1protein, peptides and analogs thereof into particles of a polymericmaterial such as polyesters, polyamino acids, hydrogels, poly(lacticacid) or ethylene vinylacetate copolymers. Alternatively, instead ofincorporating these agents into polymeric particles, it is possible toentrap these materials in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.

When oral preparations are desired, the compositions may be combinedWith typical carriers, such as lactose, sucrose, starch, talc magnesiumstearate, crystalline cellulose, methyl cellulose, carboxymethylcellulose, glycerin, sodium alginate or gum arabic among others.

The proteins of the present invention may be supplied in the form of akit, alone, or in the form of a pharmaceutical composition as describedabove.

Vaccination can be conducted by conventional methods. For example, theimmunogen can be used in a suitable diluent such as saline or water, orcomplete or incomplete adjuvants. Further, the immunogen may or may notbe bound to a carrier to make the protein immunogenic. Examples of suchcarrier molecules include but are not limited to bovine serum albumin(BSA), keyhole limpet hemocyanin (KLH), tetanus toxoid, and the like.The immunogen also may be coupled with lipoproteins or administered inliposomal form or with adjuvants. The immunogen can be administered byany route-appropriate for antibody production such as intravenous,intraperitoneal, intramuscular, subcutaneous, and the like. Theimmunogen may be administered once or at periodic intervals until asignificant titer of anti-MART-1 immune cells or anti-MART-1 antibody isproduced. The presence of anti-MART-1 immune cells may be assessed bymeasuring the frequency of precursor CTL (cytoxic T-lymphocytes) againstMART-1 antigen prior to and after immunization by a CTL precursoranalysis assay (Coulie, P. et al., (1992) International Journal OfCancer 50:289-297). The antibody may be detected in the serum using theimmunoassay described above.

The administration of the vaccine or immunogen of the present inventionmay be for either a prophylactic or therapeutic purpose. When providedprophylactically, the immunogen is provided in advance of any evidenceor in advance of any symptom due to melanoma. The prophylacticadministration of the immunogen serves to prevent or attenuate melanomain a mammal. When provided therapeutically, the immunogen is provided at(or shortly after) the onset of the disease or at the onset of anysymptom of the disease. The therapeutic administration of the immunogenserves to attenuate the disease.

A preferred embodiment is a vaccine prepared using recombinant MART-1protein or peptide expression vectors. To provide a vaccine to anindividual a genetic sequence which encodes for all or part of theMART-1 nucleic acid sequence is inserted into a expression vector, asdescribed above, and introduced into the mammal to be immunized.Examples of vectors that may be used in the aforementioned vaccinesinclude, but are not limited to, defective retroviral vectors,adenoviral vectors vaccinia viral vectors, fowl pox viral vectors, orother viral vectors (Mulligan, R. C., (1993) Science 260:926-932). Theviral vectors carrying all or part of the MART-1 nucleic sequence can beintroduced into a mammal either prior to any evidence of melanoma or tomediate regression of the disease in a mammal afflicted with melanoma.Examples of methods for administering the viral vector into the mammalsinclude, but are not limited to, exposure of cells to the virus ex vivo,or injection of the retrovirus or a producer cell line of the virus intothe affected tissue or intravenous administration of the virus.Alternatively the viral vector carrying all or part of the MART-1nucleic acid sequence may be administered locally by direct injectioninto the melanoma lesion or topical application in a pharmaceuticallyacceptable carrier. The quantity of viral vector, carrying all or partof the MART-1 nucleic acid sequence, to be administered is based on thetiter of virus particles. A preferred range of the immunogen to beadministered may be about 10⁶ to about 10¹¹ virus particles per mammal,preferably a human. After immunization the efficacy of the vaccine canbe assessed by production of antibodies or immune cells that recognizethe antigen, as assessed by specific lytic activity or specific cytokineproduction or by tumor regression. One skilled in the art would know theconventional methods to assess the aforementioned parameters. If themammal to be immunized is already afflicted with melanoma or metastaticmelanoma the vaccine can be administered in conjunction with othertherapeutic treatments. Examples of other therapeutic treatmentsincludes, but are not limited to, adoptive T cell immunotherapy,coadministration of cytokines or other therapeutic drugs for melanoma.

Alternatively all or parts thereof of a substantially or partiallypurified the MART-1 protein may be administered as a vaccine in apharmaceutically acceptable carrier. Ranges of MART-1 protein that maybe administered are about 0.001 to about 100 mg per patient, preferreddoses are about 0.01 to about 100 mg per patient. In a preferredembodiment, the MART-1 peptide AAGIGILTV (SEQ. ID NO. 4) (presented insingle letter code) or analogs thereof is administered therapeuticallyor prophylactically to a mammal in need of such treatment.Alternatively, modified MART-1 peptides, examples of which are presentedin Table 14 may be used. Preferred doses may be about 0.001 mg to about100 mg, most preferred are about 0.01 mg to about 100 mg. The peptidemay be synthetically or recombinantly produced. Immunization is repeatedas necessary, until a sufficient titer of anti-immunogen antibody orimmune cells has been obtained.

In yet another alternative embodiment a viral vector, such as aretroviral vector, can be introduced into mammalian cells. Examples ofmammalian cells into which the retroviral vector can be introducedinclude, but are not limited to, primary mammalian cultures orcontinuous mammalian cultures, COS cells, NIH3T3, or 293 cells (ATTC#CRL 1573). The means by which the vector carrying the gene may beintroduced into a cell includes, but is not limited to, microinjection,electroporation, transfection or transfection using DEAE dextran,lipofection, calcium phosphate or other procedures known to one skilledin the art (Sambrook et al. (EDS) (1989) in “Molecular Cloning. Alaboratory manual”, Cold Spring Harbor Press Plainview, N.Y.). Themammalian cells expressing the MART-1 antigen can be administered tomammals and serve as a vaccine or immunogen. Examples of how the cellsexpressing MART-1 antigens can be administered include, but is notlimited to, intravenous, intraperitoneal or intralesional. In apreferred embodiment, the part of the MART-1 nucleic acid sequencecorresponding to the peptide AAGIGILTV (SEQ ID NO: 4) is inserted intothe MART-1 expression vector and introduced into the mammalian cells.Alternatively, a nucleic acid sequence corresponding to MART-1 peptideswhich have been modified to enhance their binding to MHC molecules maybe used. By way of example, the nucleic acid sequences encoding themodified peptides shown in Table 14 may be inserted into an expressionsvector and introduced with mammalian cells.

The vaccine formulation of the present invention comprise an immunogenthat induces an immune response directed against the melanoma associatedantigens such as the melanoma associated MART-1 antigen. The vaccineformulations may be evaluated first in animal models, initially rodents,and in nonhuman primates and finally in humans. The safety of theimmunization procedures is determined by looking for the effect ofimmunization on the general health of the immunized animal (weightchange, fever, appetite behavior etc.) and looking for pathologicalchanges on autopsies. After initial testing in animals, melanoma cancerpatients can be tested. Conventional methods would be used to evaluatethe immune response of the patient to determine the efficiency of thevaccine.

In yet another embodiment of this invention all, part, or parts of theMART-1 protein or MART-1 peptides or analogs thereof, or modified MART-1peptides or analogs thereof, may be exposed to dendritic cells culturedin vitro. The cultured dendritic cells provide a means of producingT-cell dependent antigens comprised of dendritic cell modified antigenor dendritic cells pulsed with antigen, in which the antigen isprocessed and expressed on the antigen activated dendritic cell. TheMART-1 antigen activated dendritic cells or processed dendritic cellantigens may be used as immunogens for vaccines or for the treatment ofmelanoma. The dendritic cells should be exposed to antigen forsufficient time to allow the antigens to be internalized and presentedon the dendritic cells surface. The resulting dendritic cells or thedendritic cell process antigens can than be administered to anindividual in need of therapy. Such methods are described in Steinman etal. (WO93/208185) and in Banchereau et al. (EPO Application 0563485A1)which are incorporated herein by reference.

In yet another embodiment of this invention T-cells isolated fromindividuals can be exposed to the MART-1 protein or portions thereof, orMART-1 peptides or analogs thereof or MART-1 modified peptides oranalogs thereof in vitro and then administered to a patient in need ofsuch treatment in a therapeutically effective amount. Examples of whereT-lymphocytes can be isolated, include but are not limited to,peripheral blood cells lymphocytes (PBL), lymph nodes, or tumorinfiltrating lymphocytes (TIL). Such lymphocytes can be isolated fromthe individual to be treated or from a donor by methods known in the artand cultured in vitro (Kawakami, Y. et al. (1989) J. Immunol. 142:2453-3461). Lymphocytes are cultured in media such as RPMI or RPMI 1640or AIM V for 1-10 weeks. Viability is assessed by trypan blue dyeexclusion assay. The lymphocytes are exposed to all or part of theMART-1 protein for part or all of the culture duration. In a preferredembodiment the lymphocytes are exposed to the AAGIGILTV (SEQ ID NO: 4)peptide (presented in single letter code) at a concentration of about 1to about 10 micrograms(ug)/ml per 10⁷ cells for all or part of theduration of lymphocyte culture. After being sensitized to the peptidethe T-lymphocytes are administered to the mammal in need of suchtreatment. Alternatively, the modified MART-1 peptides shown in Table 14may be exposed to lymphocytes. Examples of how these sensitized T-cellscan be administered to the mammal include but are not limited to,intravenously, intraperitoneally or intralesionally. Parameters that maybe assessed to determine the efficacy of these sensitized T-lymphocytesinclude, but are not limited to, production of immune cells in themammal being treated or tumor regression. Conventional methods are usedto assess these parameters. Such treatment can be given in conjunctionwith cytokines or gene modified cells (Rosenberg, S. A. et al. (1992)Human Gene Therapy, 3: 75-90; Rosenberg, S. A. et al. (1992) Human GeneTherapy, 3: 57-73).

In addition to use as a vaccine, the compositions can be used to prepareantibodies to MART-1 antigen, peptides or analogs thereof, or modifiedMART-1 peptides and analogs thereof. The antibodies can be used directlyas anti-melanoma agents. To prepare antibodies, a host animal isimmunized using the MART-1 protein, peptides or analogs thereof, ormodified peptides or analogs thereof as the immunogen and bound to acarrier as described above for vaccines. The host serum or plasma iscollected following an appropriate time interval to provide acomposition comprising antibodies reactive with the immunogen. The gammaglobulin fraction or the IgG antibodies can be obtained, for example, byuse of saturated ammonium sulfate or DEAE Sephadex, or other techniquesknown to those skilled in the art. The antibodies are substantially freeof many of the adverse side effects which may be associated with otheranti-cancer agents such as chemotherapy.

The antibody compositions can be made even more compatible with the hostsystem by minimizing potential adverse immune system responses. This isaccomplished by removing all or a portion of the Fc portion of a foreignspecies antibody or using an antibody of the same species as the hostanimal, for example, the use of antibodies from human/human hybridomas.Humanized antibodies (i.e., nonimmunogenic in a human) may be produced,for example, by replacing an immunogenic portion of an antibody with acorresponding, but nonimmunogenic portion (i.e., chimeric antibodies).Such chimeric antibodies may contain the reactive or antigen bindingportion of an antibody from one species and the Fc portion of anantibody (nonimmunogenic) from a different species. Examples of chimericantibodies, include but are not limited to, non-human mammal-humanchimeras, rodent-human chimeras, murine-human and rat-human chimeras(Robinson et al., International Patent Application 184,187; TaniguchiM., European Patent Application 171,496; Morrison et al., EuropeanPatent Application 173,494; Neuberger et al., PCT Application WO86/01533; Cabilly et al., 1987 Proc. Natl. Acad. Sci. USA 84:3439;Nishimura et al., 1987 Canc. Res. 47:999; Wood et al., 1985 Nature314:446; Shaw et al., 1988 J. Natl. Cancer Inst. 80: 15553, allincorporated herein by reference).

General reviews of “humanized” chimeric antibodies are provided byMorrison S., 1985 Science 229:1202 and by Oi et al., 1986 BioTechniques4:214.

Suitable “humanized” antibodies can be alternatively produced by CDR orCEA substitution (Jones et al., 1986 Nature 321:552; Verhoeyan et al.,1988 Science 239:1534; Siedleret al. 1988 J. Immunol. 141:4053, allincorporated herein by reference).

The antibodies or antigen binding fragments may also be produced bygenetic engineering. The technology for expression of both heavy andlight cain genes in E. coli is the subject the following PCT patentapplications; publication number WO 901443, WO901443, and WO 9014424 andin Huse et al., 1989 Science 246:1275-1281.

The antibodies can also be used as a means of enhancing the immuneresponse. The antibodies can be administered in amounts similar to thoseused for other therapeutic administrations of antibody. For example,pooled gamma globulin is administered at a range of about 1 mg to about100 mg per patient. Thus, antibodies reactive with the MART-1 antigencan be passively administered alone or in conjunction with otheranti-cancer therapies to a mammal afflicted-with melanoma. Examples ofanti-cancer therapies include, but are not limited to, chemotherapy,radiation therapy, adoptive immunotherapy therapy with TIL.

Alternatively, anti MART-1 antigen antibodies can be induced byadministering anti-idiotype antibodies as immunogens. Conveniently, apurified anti-MART-1 antibody preparation prepared as described above isused to induce anti-idiotype antibody in a host animal. The compositionis administered to the host animal in a suitable diluent. Followingadministration, usually repeated administration, the host producesanti-idiotype antibody. To eliminate an immunogenic response to the Fcregion, antibodies produced by the same species as the host animal canbe used or the Fc region of the administered antibodies can be removed.Following induction of anti-idiotype antibody in the host animal, serumor plasma is removed to provide an antibody composition. The compositioncan be purified as described above for anti-MART-1 antibodies, or byaffinity chromatography using anti-MART-1 antibodies bound to theaffinity matrix. The anti-idiotype antibodies produced are similar inconformation to the authentic MART-1-antigen and may be used to preparean MART-1 melanoma antigen vaccine rather than using the MART-1 protein,peptides analogs or portions thereof.

When used as a means of inducing anti-MART-1 antibodies in an animal,the manner of injecting the antibody is the same as for vaccinationpurposes, namely intramuscularly, intraperitoneally, subcutaneously,interlesionally, or the like in an effective concentration in aphysiologically suitable diluent with or without adjuvant. One-or morebooster injections may be desirable.

The MART-1 derived proteins or peptides or modified peptides of thisinvention are also intended for use in producing antiserum designed forpre- or post-disease prophylaxis. Here the MART-1 antigen, peptides oranalogs thereof, or modified MART-1 peptides or analogs thereof isformulated with a suitable adjuvant and administered by injection tohuman volunteers, according to known methods for producing humanantisera. Antibody response to the injected proteins is monitored,during a several-week period following immunization, by periodic serumsampling to detect the presence of anti-MART-1 serum antibodies, usingan immunoassay as described herein.

The antiserum from immunized individuals may be administered as aprophylactic measure for individuals who are at risk of developingmelanoma. The antiserum is also useful in treating an individualafflicted with melanoma for post-disease prophylaxis.

For both in vivo use of antibodies to MART-1 antigen and anti-idiotypeantibodies and diagnostic use, it may be preferable to use monoclonalantibodies. Monoclonal anti-MART-1 antibodies or anti-idiotypeantibodies can be produced as follows. The spleen or lymphocytes from animmunized animal are removed and immortalized or used to preparehybridomas by methods known to those skilled in the art. (Goding, J. W.1983. Monoclonal Antibodies: Principles and Practice, Pladermic Press,Inc., NY, N.Y., pp. 56-97). To produce a human-human hybridoma, a humanlymphocyte donor is selected. A donor known to have a melanoma carryingthe MART-1 antigen may serve as a suitable lymphocyte donor. Lymphocytescan be isolated from a peripheral blood sample or spleen cells may beused if the donor is subject to splenectomy. Epstein-Barr virus (EBV)can be used to immortalize human lymphocytes or a human fusion partnercan be used to produce human-human hybridomas. Primary in vitroimmunization with peptides can also be used in the generation of humanmonoclonal antibodies. Examples of preferred MART-1 peptides, but notlimited to are, AAGIGILTV (SEQ ID NO: 4), EAAGIGILTV (SEQ ID NO: 17) andAAGIGILTVI (SEQ ID NO: 18) (peptides are presented in single letteramino acid code). Most preferably AAGIGILTV (SEQ ID NO: 4) is used asthe immunogen. Alternatively, peptides derived from the MART-1 aminoacid sequence and modified to enhance binding of the peptide to a MHCClass I molecule may also be used. By way of example the modifiedpeptides shown in Table 14 may be used as the immunogen.

Antibodies secreted by the immortalized cells are screened to determinethe clones that secrete antibodies of the desired specificity. Formonoclonal MART-1 antigen or peptide antibodies, the antibodies mustbind to MART-1 antigen or peptide. For monoclonal anti-idiotypeantibodies, the antibodies must bind to anti-MART-1 antibodies. Cellsproducing antibodies of the desired specificity are selected.

The antibodies or chimeric antibodies described herein may also becoupled to toxin molecules radio-isotopes and drugs by conventionalmethods (Vitetta et al. (1991) in “Biologic Therapy of Cancer” De Vita VT, Hellman S., Rosenberg, S. A. (eds) J.B. Lippincott Co. Philadelphia;Larson, S. M. et al. (1991) in “Biological Therapy of Cancer” De Vita V.T., Hellman S., Rosenberg, S. A. (eds) J.B. Lippincott Co.,Philadelphia). Examples of toxins to which the antibodies may be coupledto include, but are not limited to, ricin or diphtheria toxin. Examplesof drugs or chemotherapeutic agents include, but are not limited to,cyclophosphamide or doxorubcin. Examples of radioisotopes, include, butare not limited to, ¹³¹I, Antibodies covalently conjugated to theaforementioned agents can be used in cancer immunotherapy for treatingmelanoma.

Local administration to the afflicted site may be accomplished throughmeans known in the art, including, but not limited to, topicalapplication, injection, and implantation of a porous device containingcells recombinantly expressing the infusion, implantation of a porousdevice in which the MART-1 antibodies or chimeric antibodies, antibodiescoupled to toxins, drugs or radiolabels or portions thereof arecontained.

The above described antibodies and antigen binding fragments thereof maybe supplied in kit form alone, or as a pharmaceutical composition for invivo use. The antibodies may be used for therapeutic uses, diagnosticuse in immunoassays or as an immunoaffinity agent to purify the MART-1protein or peptides as described herein.

The present invention also provides a substantially purified andisolated nucleic acid sequence, designated c(complementary)DNA25 (FIG.4; SEQ. ID. NO. 26) which encodes a second melanoma recognized by tumorinfiltrating lymphocytes. The TIL which recognize the melanoma antigenencoded by cDNA25 are associated with in vivo tumor rejection. The TILrecognized the melanoma antigen encoded by cDNA25 in the context ofHLA-A2. Comparison of the cDNA25 nucleic acid sequence (FIG. 4; SEQ. IDNO 26) with the nucleic acid sequences for genes encoding amelanocyte-melanoma specific protein gp100 shows this sequence to besimilar, but distinct, from the previously identified sequences forgp100. Previously identified sequences for gp100 include gp100 (GenBankAccession No. M32295; also designated gp95), Pmel 17 (GenBank AccessionNo. M77348; Kwon et al., (1991) Proc. Natl. Acad. Sciences (USA)88:9228-9232) and ME20 (Maresh et al. (1994) DNA and Cell Biology13:87-95).

The cDNA25 sequence provided herein (FIG. 4; SEQ. ID. NO. 26) differsfrom the previously reported gp100 sequence in Genbank (GenbankAccession No. M32295) by two nucleotides, from the Pmel 17 sequence(Kwon et al. (1991) Proc. Natl. Acad. Sciences (USA) 88: 9228-9232) bythree nucleotides and a twenty one base pair deletion, and from the ME20sequence (Maresh et al. (1994) DNA and Cell Biology 13:87-95) by asingle nucleotide difference. At the amino acid level, the proteinencoded by cDNA25 differs from the gp100 in GenBank (GenBank Accession#M32295) by one amino acid at position 162, by a two amino aciddifference at positions 162 and 274, compared to Pmel 17 and did notcontain 7 amino acids that existed in Pmel 17 at positions 588-594.Therefore, cDNA25 appears to encode for a different form of the gene forgp100. The differences between the cDNA25 nucleic acid sequence (FIG. 4;SEQ. ID. NO. 26) and amino acid sequence (FIG. 5A; SEQ. ID. NO. 27) andpreviously reported gp100 sequences may be due to polymorphisms, allelicvariations, or to mutations within the tumor. Experiments with mousetumors have shown that new antigens recognized by T-cells can resultfrom point mutation in the coding region of the inactive gene (Boon, T(1992) Advances in Cancer Research 58:177-210).

This invention also provides immunogenic peptides derived from the gp100protein sequence provided herein. (FIG. 5A; SEQ. ID. NO. 27). Theseimmunogenic peptides represent the antigenic portions of the gp100protein (FIG. 5A; SEQ. ID. NO. 27) recognized by TIL. Examples ofimmunogenic peptides include, but are not limited to, LLDGTATLRL(peptide G10-4; SEQ ID NO: 33; FIG. 4, nucleotides 1407-1436),VLYRYGSFSV (peptide G10-5; SEQ ID NO: 34; FIG. 4 nucleotides 1464-1493),ALDGGNKHFL (peptide G10-22; SEQ ID NO: 35; FIG. 4, nucleotides 708-737),VLKRCLLHL (peptide G9-19 SEQ ID NO: 36; FIG. 4, nucleotides 48-74),VLPSPACQLV (peptide G10-8; SEQ ID NO: 37; FIG. 4 nucleotides 1668 to1797) SLADTNSLAV (peptide G10-9; SEQ ID NO: 38; FIG. 4 nucleic acids1746 to 1775) SVSVSQLRA (peptide G9-216; SEQ ID NO:39; FIG. 4,nucleotides 684 to 710) YLEPGPVTA (peptide G9-280; SEQ ID NO:40; FIG. 4;nucleotides 876-902) or LNVSLADTN (peptide G10-400; SEQ ID NO:41; FIG.4; nucleotides 1736-1764) KTWGQYWQV (peptide G9₁₅₄; SEQ ID NO:46; FIG.5A; amino acids 154 to 162), KTWGQYWQVL (peptide G10₁₅₄ SEQ ID NO:47;FIG. 5A; amino acids 154 to 163), ITDQVPFSV (peptide G9₂₀₉, SEQ IDNO:48; FIG. 5A; amino acids 209 to 217) and TITDQVPFSV (peptide G10₂₀₈;SEQ ID NO: 49; FIG. 5A; amino acids 208 to 217). This invention furtherincludes analogs of these immunogenic peptides derived from the gp100amino acid sequence (FIG. 5A; SEQ ID NO: 27). The term analog includesany peptide which displays the functional aspects of these immunogenicpeptides. The term analog also includes conservative substitution orchemical derivative of the peptides as described above. Theseimmunogenic peptides may be synthetically or recombinantly produced inthe same manner or fashion as described above for MART-1.

In yet another embodiment of this invention, immunogenic peptidesderived from the gp100 sequence (FIG. 5A; SEQ ID NO: 27, are modified toincrease immunogenicity by enhancing the binding of the peptide to MHCmolecule with which the peptide is associated when presented to T-cells.By way of example, modifications may include the substitution, deletionor addition, of one or more amino acids within the immunogenic peptidesequence, or insertion of amino acids within the given immunogenicpeptide sequence or derivitization of existing amino acids within thegiven immunogenic peptide sequence or mutation of the amino acids withinthe given immunogenic peptide sequence. In a preferred modification atleast one amino acid is substituted or replaced in the given immunogenicpeptide sequence. Any amino acid composing the given immunogenic peptidesequence may be modified in accordance with this invention. Any aminoacid may be used to substitute or replace a given amino acid within theimmunogenic peptide sequence. Modification may occur at any amino acidposition within the immunogenic gp100 peptide. Modified gp100 peptidesis intended to include any modified immunogenic gp100 peptide exhibitingenhanced binding with the MHC molecule with which it is associated whenpresented to the T-cell.

By way of example peptides recognized by T cells in the context ofHLA-A2 are 9 to 10 amino acids in length. Preferably for enhancedbinding of the peptide to HLA-A2 the second position and last positionin the peptide are hydrophobic amino acids preferably aliphatichydrophobic amino acids. The second position may be any aliphatichydrophobic amino acid such as, but not limited to, leucine, methionine,isoleucine, valine, threonine, glycine or alanine. The last position ofthe peptide (position 9 or 10 depending on the peptide's length) may beany aliphatic hydrophobic amino acid, such as but not limited to valine,leucine, alanine, leucine, isoleucine, glycine, methionine, valine, orthreonine.

The first and third positions of the immunogenic peptide may besubstituted or replaced with any amino acid, preferably, hydrophobicaliphatic amino acids, or aromatic amino acids. Examples of amino acidsthat may be used at the first or third position of the peptide include,but are not limited to, alanine, leucine, lysine, isoleucine, glycine,methionine, valine, threonine, tryptophan, phenylalanine, serine, lysineor tyrosine.

Examples of gp100 peptides that may be modified in accordance with thepresent embodiment include, but is not limited to LLDGTATLRL (peptideG10-4; SEQ ID NO: 33; FIG. 4, nucleotides 1407-1436), VLYRYGSFSV(peptide G10-5; SEQ ID NO: 34; FIG. 4 nucleotides 1464-1493), ALDGGNKHFL(peptide G10-22; SEQ ID NO: 35; FIG. 4, nucleotides 708-737), VLKRCLLHL(peptide G9-19 SEQ ID NO: 36; FIG. 4, nucleotides 48-74), VLPSPACQLV(peptide G10-8; SEQ ID NO: 37; FIG. 4 nucleotides 1668 to 1797)SLADTNSLAV (peptide G10-9; SEQ ID NO: 38; FIG. 4 nucleic acids 1746 to1775) SVSVSQLRA (peptide G10-216; SEQ ID NO:39; FIG. 4, nucleotides 684to 710) YLEPGPVTA (peptide G9-280; SEQ ID NO:40; FIG. 4; nucleotides876-902) or LNVSLADTN (peptide G10-400; SEQ ID NO:41; FIG. 4;nucleotides 1736-1764) KTWGQYWQV (peptide G9₁₅₄; SEQ ID NO:46; FIG. 5A;amino acids 154 to 162), KTWGQYWQVL (peptide G10₁₅₄ SEQ ID NO:47; FIG.5A; amino acids 154 to 163), ITDQVPFSV (peptide G9₂₀₉, SEQ ID NO:48;FIG. 5A; amino acids 209 to 217) and TITDQVPFSV (peptide G10₂₀₈; SEQ IDNO: 49; FIG. 5A; amino acids 208 to 217).

By way of example modified gp100 peptides derived from the immunogenicgp100 peptide KTWGQYWQV (SEQ ID NO:46) may have the formulaX₁X₂X₃GQYWQX₄ wherein:

X₁ may be any amino acid, preferably any hydrophobic aliphatic aminoacid, or aromatic amino acid. Examples of amino acids that may be usedinclude, but are not limited to, alanine, leucine, lysine, isoleucine,glycine, methionine, valine, threonine, tryptophan, phenylalanine,lysine or serine, aspartic acid or tyrosine;

X₂ may be any hydrophobic amino acid, preferably any aliphatichydrophobic amino acid. Examples of amino acids that may be usedinclude, but are not limited to, leucine, methionine, isoleucine,alanine, threonine, glycine, or valine. Most preferably leucine,methionine or isoleucine.

X₃ may be any amino acid, preferably any hydrophobic aliphatic aminoacid or aromatic amino acid. Examples of amino acids that may be usedinclude, but are not limited to, alanine, leucine, lysine, isoleucine,glycine, methianine, valine, threonine, tryptophan, phenylalanine,serine, lysine, or tyrosine;

X₄ is any hydrophobic amino acid, preferably an aliphatic hydrophobicamino acid. Examples of amino acids that may be used include, but arenot limited to, valine, leucine, isoleucine, methionine, alanine,threonine, or glycine.

Examples of modified peptides are shown in Table 15. A preferredmodified peptide is KIWGQYWQV (G9-154-2I).

Alternatively, the immunogenic gp100 ITDQVPFSV (G9-209; SEQ ID NO:48)may be modified, such modified peptides may have the general formulaX₁X₂X₃QVPFSX₄ wherein:

X₁ may be any amino acid, preferably any hydrophobic aliphatic aminoacid, or aromatic amino acid. Examples of amino acids that may be usedinclude, but are not limited to, leucine, methionine, alanine,isoleucine, valine, threanine, glycine, lysine, phenylalanine,tryptophan, or tyrosine, aspartic acid or serine;

X₂ is any hydrophobic amino acid, preferably a hydrophobic aliphaticamino acid. Examples of amino acids that may be used include, but arenot limited to, leucine, methionine, alanine, isoleucine, valine,threanine, or glycine;

X₃ may be any amino acid, preferably any hydrophobic, aliphatic aminoacid or aromatic amino acid. Examples of amino acids that may be usedinclude, but are not limited to, leucine, methionine, alanine,isoleucine, valine, threonine, glycine, lysine, phenylalanine,tryptophan, tyrosine, aspartic acid or serine;

X₄ may be any hydrophobic amino acid, preferably any hydrophobicaliphatic amino acid. Examples of amino acids that may be used include,but are not limited to, leucine, methionine, alanine, isoleucine,valine, or threonine;

Examples of modified peptides derived from ITDQVPFSV are shown in Table16. Preferably the peptide FLDQVPFSV (peptide G9-209-1F2L) is used.

By way of example modified gp100 peptides derived from the immunogenicgp100 peptide YLEPGPVTA (G9-280; SEQ ID NO:40) may also be modified toenhance binding to MHC Class I molecules, preferably HLA-A2 or subtypesthereof.

The modified peptides may have the general formula X₁X₂X₃PGPVTX₄wherein:

X₁ may be any amino acid, preferably a hydrophobic aliphatic amino acidor aromatic amino acid. Examples of amino acids that may be usedinclude, but are not limited to, leucine, methionine, alanine,isoleucine, valine, threanine, glycine, lysine, phenylalanine,tryptophan, or tyrosine, aspartic acid or serine;

-   -   X₂ may be any hydrophobic amino acid, preferably an aliphatic        hydrophobic amino acid. Examples of amino acids that may be used        include, but are not limited to leucine, methionine, alanine,        isoleucine, valine, threonine, or glycine;

X₃ may be any amino acid, preferably any hydrophobic aliphatic aminoacid, or aromatic amino acid. Examples of amino acids that may be usedinclude, but are not limited to, leucine, methionine, alanine,isoleucine, valine, threanine, glycine, lysine, phenylalanine,tryptophan, tyrosine, aspartic acid or serine;

X₄ may be any hydrophobic amino acid preferably an aliphatic hydrophobicamino acid. Examples of amino acids that may be used include but are notlimited to, leucine, methionine, alanine, isoleucine, valine, threonine,or glycine.

Examples of modified peptides derived from YLEPGPVTA (G9-280; SEQ IDNO:40) are shown in Table 17. A preferred modified peptide is YLEPGPVT(G9-280-9V).

This invention further includes analogs of these modified peptidesderived from the gp-100 sequence (FIG. 5A; SEQ ID NO:27). The termanalog is intended to include any peptide which displays the functionalaspects of these modified peptides as described above. These modifiedpeptides may be synthetically or recombinantly provided by conventionalmethods.

In another embodiment the immunogenic peptides (SEQ ID NO: 33 to 38 and46 to 49) derived from the gp100 amino acid sequence (SEQ ID NO: 27) ormodified gp100 peptides as shown in Tables 15-17 or analogs thereof, maybe used as a vaccine either therapeutically or prophylactically. Whenprovided, prophylactically the vaccine is provided in advance of anyevidence of melanoma. The prophylactic administration of these peptidesshould serve to prevent or attenuate melanoma in a mammal.

In a preferred embodiment, mammals, preferably humans, at high risk formelanoma are prophylactically treated with these vaccines.Alternatively, the vaccine may be provided therapeutically to enhancethe patients own immune response to the tumor antigen prescribed on themelanoma or metastatic melanoma. The vaccine, which acts as animmunogen, may be a cell, cell lysate from cells transfected with arecombinant expression vector carrying a nucleic acid sequences encodinggp100 immunogenic peptide or a culture supernatant containing theexpressed protein. Expression vectors into which nucleic acid sequencesencoding these immunogenic peptides may be introduced are the same asthose described above for MART-1. Alternatively, the immunogen is apartially or substantially purified recombinant gp100 peptide or analogthereof.

While it is possible for the immunogen to be administered in a pure orsubstantially pure form, it is preferable to present it aspharmaceutical compositions, formulations or preparations as describedabove for MART-1. Vaccination can be conducted by conventional methodspreviously described above for MART-1.

The gp100 immunogenic peptides and nucleic acids sequences encoding themmay be used in bioassays, or to generate antibodies in the same manneror fashion as described above for MART-1.

In yet another embodiment of this invention, multivalent vaccinesagainst one or more melanoma antigens are provided. Such multivalentvaccines may comprise all or part of the MART-1 protein peptides ormodified peptides or gp100 peptides or modified peptides or combinationsthereof.

Previous identification of genes encoding melanoma antigens haveutilized PBL isolated from melanoma patients immunized or pretreatedwith antigens (Van Der Bruggen et al. (1991) Science 254: 1643-1647;Brichard et al. (1993) J. Exp. Med. 178: 489-495; Traversari, C., et al.(1992) J. Exp. Med. 176: 1453-1457). A preferred strategy is to identifygenes coding for tumor antigens that are recognized by TIL fromtumor-bearing patients, in the absence of immunization of said patients.Such a strategy enhances the possibility that the genes identified codefor antigens involved in the natural immune response against the growingcancer. Thus, this invention also provides a method of identifying genesencoding melanoma antigens utilizing cDNA expression cloning usingtumors infiltrating lymphocytes isolated (TIL) from the tumor ofpatients afflicted with melanoma. The method comprises the followingsteps: (a) isolating tumor infiltrating lymphocytes from the tumor of amammal afflicted with melanoma; (b) introducing a melanoma cDNA libraryinto a mammalian cell line; (c) exposing said mammalian cells to saidTIL; (d) screening for expression of an antigen encoded by said cDNA insaid mammalian cells recognized by said TIL; and (e) isolating said cDNAcorresponding to said antigen. The tumor infiltrating lymphocytes instep (a) may be isolated from patients afflicted with melanomaincluding, but not limited to, the melanoma lesion, subcutaneous tissueor visceral organs. Examples of cells that may be used to prepare thecDNA library used in step (b), include, but are not limited to, fresh orcultured melanoma cells. Preferably, the cDNA library is introduced intomammalian cells not expressing melanoma antigens. If non human mammaliancells or human cells not expressing the desired HLA haplotype forrecognition by the TIL are used in step (b), such cells can becotransfected with an HLA gene as described below. Examples of cellswhich can be used in step (b), include but are not limited to, tumorcell lines, such as breast cancer cell line MDA 231 (ATCC #HTB26), orCOS 7 cells (ATCC #CRL 1651). Examples of MHC genes which can be usedinclude, but are not limited to, HLA-A, HLA-B, and HLA-C genes,preferably HLA-A2 and subtypes thereof (Zemmour, J. et al. (1992) TissueAntigens 40:221-228). The appropriate MHC gene to be used is determinedby the haplotype of the tumor cells which were the source for the cDNAlibrary. Standard methods can be used to determine the haplotyperecognized by the TIL isolated (ASHI Laboratory Manual (2nd Edition1990). Examples of how to evaluate recognition of the cells containingthe cDNA clone expressing an antigen recognized by the TIL includes, butis not limited to, γ-interferon assays, TNF secretion (Van de Bruggen etal., (1991) Science 254:1643-1647) or lysis of cells transfected withcDNA encoding for the recognized antigen. Such assays are performed byconventional methods known to one skilled in the art. Melanoma antigenscan be isolated by or rescued by PCR using primer specific to flankingsite of vector containing the cDNA. Examples of how to isolate the cDNAcorresponding to the antigen recognized by the TIL include, but are notlimited to, PCR.

Once the genes or nucleic acid sequences encoding melanoma antigens areidentified, the next step is to determine the antigenic portion orepitope of the protein encoded by these genes. Therefore, in yet anotherembodiment of this invention, a method is provided for assessing theimmunogenicity of peptides derived from the predicted amino acidsequences of the MART-1 protein (FIG. 1; SEQ ID NO: 2) or a gp100protein (FIG. 5A; SEQ ID NO: 27). The method comprises the steps of: (a)preparing a plurality of peptides based on the MART-1 (FIG. 1; SEQ IDNO: 2) or gp100 (FIG. 5A; SEQ ID NO: 27) amino acid sequence; (b)incubating at least one of said peptides with a mammalian cell line; (c)exposing said mammalian cells incubated with said peptide to tumorinfiltrating lymphocytes (TIL); and (d) screening for recognition of TILwith said cells incubated with said peptide. It is preferred thatpeptides of about 25 to 5 amino acids be used, more preferably 20 to 10amino acids and most preferably 9-10 amino acids. Examples of cells thatmay be used in step (b) include, but are not limited to, T2-cells,(Cerundolo, V. et al. (1990) Nature, 345: 449-452) or EBV transformed Bcell lines (Topalian et al. (1989) J. Immunol. 142: 3714-3725). Examplesof how to assess recognition of the cells incubated with peptide includebut is not limited to, ⁵¹CR release cytotoxicity assay (Cerundolo, V. etal. (1990) Nature 345:449-452.) or lymphokine assays such as γ-IFN orTNF secretion. (Schwartzentruber, D. et al., (1991) J. of Immunology146:3674-3681).

T cells recognize antigen complexed with MHC Class 1 molecules. The MHClocus in all mammalian species contains numerous genes and is highlypolymorphic. Different MHC molecules or haplotypes types bind differentantigens. In humans the HLA complex contains the HLA-A, HLA-B and HLA-Cgene loci which encode class I molecules. Lymphocytes will recognizetumor antigens on the context of HLA Class 1 molecule. If the cellscontaining the recombinant MART-1 expression vector are to be screenedby the TIL but are not human cells, such as COS cells, or do not expressa desired haplotype an expression vector containing an MHC Class 1 genemay also be introduced into the cells. (See Example 1) This, representsyet another alternative embodiment of the invention. Cells expressingMART-1 antigens and HLA antigens can by screened with TIL to detect thepresence of tumor antigens in the context of a specific MHC Class 1restriction type. The appropriate haplotype is determined by thehaplotype of the tumor from which the library is derived. Examples ofMHC Class I genes that may be used include, but are not limited to,HLA-A, HLA-B and HLA-C genes. Examples of preferred MHC specificities orrestriction types include, but is not limited to HLA-A1, HLA-A2, such asthe HLA-A2.1 subtype, or HLA-A24 (Zemmour, J. et al. (1992) TissueAntigens 40:221-228). Most preferred is the HLA-A2.1 gene.

Veterinary uses are also intended to be encompassed bit the compositionsand therapeutic applications described herein.

All books, articles, and patents referenced herein are incorporated byreference in their entirety. The following examples illustrate variousaspects of the invention and in no way intended to limit the scopethereof.

Cloning of a Gene Coding for a Shared Human Melanoma Antigen Recognizedby Autologous T Cells Infiltrating into Tumors EXAMPLE 1 Generation ofCytotoxic T Lymphocytes (CTL) and Culture of Cell Lines

−54 CTL were generated from excised tumor specimens by culturing asuspension of cells with 6000 IU/ml of IL-2 (Cetus-Oncology Division,Chiron Corp. Emeryville, Calif.) for 30-70 days as described inKawakami, Y., et al. (1988) J. Exp. Med. 168:2183-2191. TIL501 andTIL1235 were predominantly CD8⁺ and were derived from the tumorspecimens of patients with advanced metastatic melanoma. The CD8⁺ T cellclone, TIL501.A42, was established by limiting dilution methods andcultured with 120 IU/ml of IL-2 plus irradiated (once a week for 4 to 6times) autologous tumor cells.

Melanoma-cell lines, 397mel, 501mel, 526mel, 537mel, 624mel, 888mel,952mel, and Epstein-Barr virus (EBV) transformed B cell lines, 501EBVB,836EBVB were established in our laboratory and cultured in RPMI1640(GIBCO/Lifetechnologies, Grand Island N.Y.) medium containing 10% fetalcalf serum (FCS) (Biofluids, Rockville Md.). (Topalian et al., (1989) J.Immunol. 142: 3714-3725) Normal cultured melanocytes, NHEM483, NHEM493,NHEM527, NHEM529, NHEM530, NHEM533, NHEM616 and NHEM680 were purchasedfrom Clonetics, San Diego, Calif., FM725, FM801, FM902 were provided byM. Herlyn, Wistar Institute, Philadelphia Pa., HA002 was provided by R.Halaban, Yale university, New Haven, Conn. and cultured in melanocytegrowth medium (MGM, Clonetics). Melanoma cell lines, C32, RPMI7951,WM115, A375, HS695T, Malme3M, colon cancer cell lines, Collo, SW480,WiDr, breast cancer cell lines, MDA231, MCF7, HS578, ZR75, neuroblastomacell line, SK—N—SH, glioma cell lines, U138MG, HS683, H4, sarcoma cellline 143B, embryonal kidney cell line 293 transformed with adenovirustype 5 were purchased from ATCC, Rockville, Md. Renal cancer cell lines,UBK108 and UOK117 were provided M. Linehan NIH, Bethesda, Md. The smallcell lung cancer cell line, H1092 was provided by J. D. Minna,University Texas Southwestern, Dallas, Tex. Ewing's sarcoma cell lines,TC71, RD-ES, 6647 were provided by M. Tsokos, NIH, Bethesda, Md. Theneuroblastoma cell line, SK—N-AS was provided by O. M. El Badry, NIH,Bethesda, Md. The plasmacytoma cell line, HMY-C1R and the M1 fibroblastcell line were provided by W. Biddison, NIH, Bethesda, Md. Kidneyepithelial cells, KAM, WLC were provided by D. J. Hazen-Martin and D. A.Sens, Medical University of South Carolina, Charleston, S.C. The monkeykidney cell line, COS7, was provided by W. Leonard, NIH, Bethesda, Md.

Cytotoxicity Assay

⁵¹Cr release assays were performed as described in Kawakami, Y., et al.,(1988) J. Exp. Med. 168:2183-2191. Briefly, 5000 target cells labeledwith ⁵¹Cr were mixed with varying numbers of effector cells andincubated for 5 hours (h). Then supernatants were collected,radioactivity was measured and the percent specific lysis wascalculated.

IFN-γ Release Assay

Fifty to one hundred thousand responder cells and 4×10⁴-10⁵ stimulatorcells were mixed in 300 ul of AIM-V medium containing 120 IU/ml IL-2 perwell in a 96 flat-well microplate. After incubation for 20 h, 100 ul ofsupernatants were collected and added to an enzyme-linked immunosorbentassay (ELISA) plate (Immunoplate MaxiSorp, Nunc, Denmark) coated withanti-human IFN-γ monoclonal antibody (mAb) (Biosource, Camerillo,Calif.). After overnight incubation at 4° C., the plates were washed 3times and 100 ul of a 1:2000 dilution of rabbit anti-human IFN-γpolyclonal antibody (Ab)(Biosource, Camerillo, Calif.) was added andincubated at 37° C. for 2 h. Plates were washed 3 times, and 100 ul of a1:2000 dilution of alkaline phosphatase labelled goat anti-rabbit IgGpolyclonal antibody (Ab) (Boehringer Mannheim, Indianapolis, Ind.) wasadded. After a 1 h incubation at 37° C., 100 ul of 4 mg/ml p-Nitrophenylphosphate (Sigma, St Louis, Mo.) was added, incubated for 10-20 min atroom temperature in the dark, and 25 ul of 1N NaOH was added to stop thereaction. Optical density was measured at 405 nm wave length and theconcentration of IFN-γ was calculated in comparison to recombinant IFN-γstandards (Biogen, Cambridge, Mass.) measured in the same assay.

cDNA Expression Cloning

A cDNA library was constructed from poly A RNA from the HLA-A2⁺ melanomacell line, 501mel as described in (Miki, T., et al., (1989) Gene;83:137-146 Miki et al. (1991) Proc. Nat. Acad. Sci. USA 88:5167-5171).Briefly, first strand cDNA was synthesized with a linker primerGGACAGGCCGAGGCGGCC(T)₄₀ followed by second strand cDNA synthesis. Aftertreatment with T4 DNA ligase, an SfiI adaptor consisting of twooligonucleotides, CCAATCGCGACC and GGTCGCGATTGGTAA was ligated to theend of the cDNA. The cDNA was digested with SfiI and the digestedfragment was isolated by passing through a spun column. The cDNA wasthen mixed with bacteriophage λpCEV27 (Miki, T. et al., (1991) Proc.Natl. Acad. Science (USA) 88: 5167-5771) vector arms prepared by SfiIdigestion and in vitro packaging was performed.

To screen for melanoma antigens, 10 ug of the amplified cDNA librarycontaining approximately 10⁷ clones was transfected into the HLA-A2⁺antigen non-expressing cell lines, MDA231 clone 7 and A375 clone 1-4using a modified calcium-phosphate method (Mammalian Transfection Kit,Stratagene). After G418 (BRL, Gaithersburg, Md.) selection, individualcolonies were isolated and cultured in 96 well microplates and replicaplates were made. A mixture of 5×10⁴ TIL1200 and 5×10⁴ TTL1235 was addedto the wells of the microplates containing the growing transfectantsthat were near confluence and incubated for 20 h. Supernatants werecollected and IFN-γ was measured by ELISA.

Polymerase chain reaction(PCR) was performed to rescue the transfectedgenes from the genomic DNA of positive transfectants using SP6 and T7primers which flank the inserted genes. The amplified products werecloned in the pCRII vector (Invitrogen, San Diego, Calif.). For cDNAclones 22 and 23, a Hind III and XhoI fragment containing the fulllength cDNA was subcloned into the expression vector pcDNA3 (Invitrogen,San Diego, Calif.).

To test whether the cloned cDNAs encode tumor antigens, the pcDNA3containing the cloned genes were transiently transfected into the COS7cell line by the DEAE dextran method (Seed, B. and Aruffo, A. (1987)Proc. Natl. Acad. Sci. (USA) 84:3365-3369). Briefly, 3×10⁵ cells perwell in 6 well plates were incubated at 37° C. for 4 hours (h) in 0.75ml DMEM containing 10 ug of DEAE dextran (Sigma), 0.1 mM chloroquine andlug of the pcDNA3 containing the cloned genes and/or the pcDNA-HLA-A2.1(Zemmour, J. et al. (1992) Tissue Antigens 40: 221-228). After mediumwas removed, 10% DMSO solution in HBSS buffer was added and incubatedfor 2 min. The cells were washed once with PBS and incubated in 7.5% FCSDMEM for 2 days. The 293 cell line was transiently transfected usinglipofectamine (BRL, Gaithersburg, Md.) according to the manufacturer'srecommendation. After incubation, the ability of the transfected COS7 or293 cells to mediate IFN-γ release from TIL was assessed. The expressionof the HLA-A2 gene was tested by flow cytometry. Stable transfectantswere made by the calcium-phosphate method and individual colonies andpooled transfectants were tested for reactivity to TIL by cytotoxicityand IFN-γ release assays.

DNA sequencing of the cloned genes was performed by dideoxy chaintermination method with dGTP and 7-deaza-dGTP. DNA and protein sequenceswere analyzed by the GCG program with GeneBank, and EMBL Data Librarynucleotide databases and SWISS-PROT, PIR, GenPept, Brookhaven ProteinData Bank protein databases.

Northern Blot Analysis

Total RNA was isolated by the guanidine-isothiocyanate-cesium chloridecentrifugation method. (Chirgwin, J. M. et al. (1979) Biochemistry 18:5294). Total RNA from normal tissue was purchased from Clontech, (PaloAlto, Calif.). Ten to twenty micrograms of total RNA was subjected toelectrophoresis in a 1% agarose formaldehyde gel and transferred to anylon membrane (Duralon-UV membranes, Stratagene, La Jolla, Calif.) TheSal I digested fragment containing the full length cDNA from clone 22and the β-actin cDNA (Clontech) were labeled by random priming and usedas a probe. Hybridization with the probe was performed according to theQuikHyb protocol (Stratagene) at 68 C for 2-16 h. Membranes were washedtwo times with 2×SSC/0.1% SDS at 60 C for 15 minutes (min) and once with0.1×SSC at 60 C at for 30 min and then autoradiography was performed.

Characterization of Cultured TIL from Melanoma Patients

Multiple TIL lines were established from HLA-A2⁺ melanoma patients andtested for lysis of melanoma cell lines from HLA-A2⁺ and HLA-A2⁻patients. HLA typing of patients is performed by conventional HLA typingtechniques. HLA-A2 was selected because it is the most frequentlyexpressed class I MHC antigen (about 50% of individuals) and has beenshown to be a dominant restriction element for the recognition ofmelanoma antigens (Crowley, N. J., et al. (1991), J. Immunol.146:1692-1694). TIL501, TIL1235 and TIL1200 exhibited specificrecognition of shared melanoma antigens in an HLA-A2 restricted fashion.TIL501.A42 was a T cell clone established from TIL501 by limitingdilution. These TIL caused lysis or released cytokines including IFN-γ,TNFα and GM-CSF when cocultured with a variety of HLA-A2⁺ melanoma ormelanocyte cell lines but not HLA-A2⁻ melanoma lines or HLA-A2⁺non-melanoma cell lines including the breast cancer cell line, MDA 231.Two representative experiments are shown in Table 1. Thus, these CTLseemed to recognize a non-mutated peptide derived from a melanocytelineage specific antigen.

Cloning of cDNA Coding for Melanoma Antigens Recognized by T Cells

A cDNA library from the HLA-A2⁺ 501mel melanoma cell line wastransfected into two highly transfectable HLA-A2.1⁺ cancer cell lines,MDA231 and A375. These cell lines were not lysed by melanoma specificTIL but were lysed by HLA-A2 restricted influenza M1 specific CTL afterincubation with the M1₅₈₋₆₆ peptide (GILGFVFTL; single letter code)derived the influenze matrix protein or infection with a recombinantvaccinia virus containing the M1 gene (data not shown). Thus, these celllines exhibited normal antigen processing and presenting ability butwere not lysed by these melanoma-specific TIL because of the lack ofexpression of the relevant melanoma antigens. After selection with G418,approximately 6700 transfected clones from each cell line were isolatedand grown in microplates. Using the IFN-γ release assay, 21 MDA231 and27 A375 positive clones were isolated and rescreened. Of these clones,eight MDA231 and seven A375 clones were positive in a second screeningassay.

In order to rescue the integrated genes, PCR using genomic DNA fromthese positive transfectants was performed with SP6 and T7 primersflanking the insert genes. Eight genes that were amplified from theseven transfectants which showed 1 to 2 sharp bands, including a 1.6 Kbband from MDA-22 and MDA-23 transfectants, were subcloned into the pCRIIcloning vector, and then further cloned into the pcDNA3 eukaryoticexpression vector. The 1.6 Kb band detected by Northern blot analysiswith the cDNA 22 probe suggested that this fragment was a full lengthcDNA.

Transient transfection of the expression vector pcDNA3 containing thecDNA from clones 22 or 23 into either COS7 or 293 cells along with theHLA-A2.1 gene conferred reactivity to TIL1235 and TIL501.A42 asdemonstrated by the specific release of IFN-γ (Table 2, Experiments 1and 2). Stable transfection of these cDNA fragments into MDA 231 orA375mel cell lines also conferred reactivity to TIL1235 and TIL501.42(Table 2, Experiment 3). TIL501.A42 could lyse MDA231 stably transfectedwith cDNA 22 (data not shown). These results indicated that these cDNAsencode a melanoma antigen recognized by HLA-A2 restricted TIL frommelanoma patients. Transfection of another clone, MDA-25 stimulated therelease of interferon-γ only from TIL 1200.

TABLE 1 Specificity of TIL 501.A42 and TIL 1235 for Melanomas andMelanocytes A. Lytic Specificity* B. Cytokine Release Specificity**TIL1235 Stimulator** TIL1235 TIL586⁺⁺ Target* HLA-A2 TIL501.A42 % LysisLAK⁺ Cells HLA-A2 TIL501.A42 pg IFN- γ/ml 501mel + 54 51 78 501mel + 647219 <50 526mel + 25 33 74 586mel − <50 <50 1034 624mel + 23 27 75NHEM493 + 1835 850 <50 952mel + 10 11 75 NHEM527 + 1638 749 <50Malme3M + 36 41 70 NHEM530 + 1224 2532 <50 C32 + 17 23 82 NHEM533 + 300251 <50 RPMI7951 + 1 6 67 NHEM616 + 635 423 <50 WM115 + −2 3 68 FM725 +5975 1471 <50 HS695T + 1 2 87 FM801 + 1375 893 62 397mel − −1 0 70NHEM483 − <50 <50 <50 MDA231 + 0 3 94 NHEM680 − <50 <50 548 HA002 − <50<50 <50 TIL 501.A42 and TIL1235 lysed most HLA-A2 melanoma cell linesand secreted IFN-γ when cultured with HLA-A2 melanomas and melanocytes.*51Cr release assay was performed at E:T = 20:1 for TIL501.A42, at 40:1for TIL1235. All targets were melanoma cell lines except for MDA231which was a breast cancer cell line. **IFN-γ in the supernatant wasmeasured after TIL and stimulator cells were coincubated for 20 hr.501mel and 586mel are melanoma cell lines. All others were normalmelanocyte cell lines. ⁺LAK: Lymphokine activated killer cells. ⁺⁺TIL586is class I MHC restricted melanoma specific TIL, not restricted byHLA-A2.

Characterization of this cDNA revealed it to be similar, but distinct,from a previously described melanoma antigen gp100 recognized bymonoclonal antibody HMB45. This clone is described in more detail inExample 3.

The cDNA sequence of clones 22 and 23 were identical except at a singlebase that was believed to be a change introduced by PCR. Two otherindependently amplified fragments were also sequenced to clarify thisregion and the consensus sequence is shown in FIG. 1. The longest openreading frame in this gene consists of 354 bases corresponding to a 118amino acid protein of 13 kd. This sequence did not show significantsimilarity to any complete nucleotide or protein sequences inestablished databases. Amino acids 27-47 consist of a hydrophobic regionthat may contain the HLA-A2 binding peptides (Falk, K., et al. (1991),Nature 351:290-296; Hunt, D. F., et al. (1992), Science 255:1261-1263;Ruppert, J., et al. (1993), Cell 74:929-937; Nijman, H. W., et al.(1993), Eur. J. Immunol. 23:1215-1219). The antigen encoded by the cDNA22 and 23, was designated the MART-1 antigen (Melanoma AntigenRecognized by T cells-1). Of the ten HLA-A2 restricted TIL linesgenerated nine recognized MART-1, and four recognized a form of gp100isolated and described herein (see Example 3) and none appeared torecognize MAGE-1 (Zakut, R., et al. (1993), Cancer Res 53:5-8.; data notshown).

TABLE 2 Interferon Gamma Secretion by TIL501.A42 and TIL 1235 WhenCultured with HLA-A2⁺ Cell Lines Transfected with the Gene 22 or 23TIL501.A42 TIL1235 Stimulator Cell Transfected Gene HLA-A2 pg/ml Exp. 1501mel none + 1009* 1076 397mel none − <50 <50 COS7 none − <50 <50 COS7HLA-A2.1 + <50 <50 COS7 22 − <50 <50 COS7 HLA-A2.1+22 + 771 1049 Exp. 2501mel none + ND⁺ 1051 397mel none − ND <50 293 HLA-A2.1 + ND <50 293 22− ND <50 293 HLA-A2.1+22 + ND 255 Exp. 3 501mel none + 1073  1056 397melnone − <50 <50 MDA231 none + <50 <50 MDA231 23 + 674 725 A375 none + <50<50 A375 23 + 264 131 IFN-γ in the supernatant was measured after TILwere coincubated for 20 hr with COS7 or 293 cell lines transientlytransfected with the pcDNA3 containing the HLA-A2.1 and/or cDNA 22 bythe DEAE-dextran method (Exp. 1 & 2), or with the A375 or MDA231 celllines stably transfected with cDNA 23 (Exp. 3). IFN-γ was secreted onlywhen TIL were incubated with HLA-A2⁺ cell lines transfected with thecDNA 22 or 23. *IFN-γ secreted by TIL alone without stimulator (<50pg/ml) was subtracted. ⁺Not done

TABLE 3 Expression of the MART-1 Gene in a Variety of Tissue and CellLines Melanoma HLA-A2+ 501mel + 526mel + 624mel + Malme3M + 952mel +697mel + C32 + RPMI7951 − WM115 − A375 − HLA-A2− 397mel + 888mel +537mel + 586mel + Melanocyte NHEM483 + NHEM493 + NHEM529 + NHEM530 +FM902 + FM906 + HA002 + Normal fresh tissue Brain − Retina + Adrenalgland − Lung − Liver − Kidney − Spleen − Thymus − Testis − Fetal liver −Non-melanoma cell lines T-cell TILA − TILB − B-cell Daudi − HMY-CIR −501EBVB − 836EBVB − Fibroblast M1 fibroblast − Kidney epithelial cellKAM − WLC − Colon cancer Collo − SW480 − WiDr − Breast cancer MDA231 −MCF7 − HS578 − ZR75 − Neuroblastoma SK-N-AS − SK-N-SH − Ewing sarcomaTC75 − RD-ES − 6647 Sarcoma 143B − Glioma U138MG − HS683 − Renal cellcancer UOK108 − UOK117 − Small cell lung cancer H1092 − Northern blotanalysis with 10-20 ug of total RNA was probed with the full length cDNAof the gene 22. The RNA from most melanomas, all melanocyte cell linestested and retina were positive.

Expression of MART-1

Northern blot analysis of a variety of cell lines including melanoma,melanocyte and nonmelanoma cancer cell lines and normal tissues wasperformed to evaluate the expression of the gene coding for MART-1(Table 3). Seven of ten HLA-A2⁺ melanoma cell lines, all four HLA-A2⁻melanoma cell lines, and all seven melanocyte cell lines tested werepositive for MART-1 RNA expression. In this Northern analysis, allHLA-A2⁺ melanoma cell lines recently established in our laboratoryexpressed MART-1 RNA. There was a perfect correlation between MART-1expression and lysis by TIL501.A42 in the 10 HLA-A2⁺ melanoma linesshown in Table 3. TIL 501.A42 which recognized the MART-1 Ag lysed 13 of17 (76%) HLA-A2⁺ melanoma cell lines tested (data not shown). Of tennormal human tissues examined for mRNA expression by Northern blotanalysis only retina was positive. No positivity was seen in any celllines from T cells, B cells, kidney epithelial cells or fibroblasts orin 19 nonmelanoma tumors. It thus appears that MART-1 is a previouslyundescribed antigen expressed on melanocyte lineage cells from skin andretina that is also expressed on melanoma cells.

Studies using a panel of T cell clones and immunoselected melanomaclones (Knuth, A., et al. (1989), Proc. Natl. Acad. Sci. (USA)86:2804-2808; Wolfel, T., et al. (1987), J. Exp. Med. 170:797-810), aswell as studies analyzing HPLC fractionated peptides from melanoma cells(Slingluff, C. L., et al. (1993), J. Immunol. 150:2955-2963; Storkus, W.J., et al. (1993), J. Immunol. 151:3719-3727) suggest that multipleantigenic peptides that can provoke an immune response exist onmelanomas. By cDNA cloning, two genes encoding melanoma antigens havebeen identified; MART-1 (FIG. 1; SEQ ID NO. 1) and a gp100 gene (seeExample 3; FIG. 4; SEQ ID NO. 26). MART-1 and the form of gp100identified herein (FIGS. 4 & 5A; SEQ. ID NOS. 26 and 27), both arerecognized by HLA-A2.1 restricted TIL. The MART-1 antigen is a 118 aminoacid protein of approximately 13 kd. Neither the gene nor the amino acidsequence for MART-1 have been previously described.

MART-1 RNA was expressed in 11 of 14 HLA-A2.1 positive or negativemelanoma lines, and 7 of 7 melanocyte lines. With the exception ofretinal tissue no MART-1 expression was found on any normal tissuetested, T-cell lines, B-cell lines, kidney epithelial lines, afibroblast line or 19 tumor cell lines from cancers of the colon,breast, brain, kidney, lung or bone.

Another melanoma antigen, MAGE-1, has been described that is recognizedby T cells derived from peripheral blood lymphocytes followingrepetitive in vivo or in vitro immunization (Van Der Bruggen, et al.(1991), Science 254:1643-1647).

The identification of genes associated with melanoma tumor antigensopens new possibilities for active specific immunization approaches tothe immunotherapy of patients with cancer based on the introduction ofthese genes into viral or bacterial vector systems. The possibilityexists that immune reactions induced against melanocyte-melanoma lineageantigens such as MART-1 may be generated against normal cells. Vitiligo,probably resulting from anti-melanocyte immune reactions, has beenreported to be associated with a favorable prognosis in patients withmelanoma (Nordlund, J. J., et al. (1983), J. Am. Acad. Dermatol.9:689-695); Bystryn, J-C, et al. (1987), Arch. Dermatol. 123:1053-1055),and has also been reported in patients responding to chemoimmunotherapy(Richards, J. M., et al. (1992), J. Clin. Oncol. 10:1338-1343). TIL withanti melanocyte-melanoma reactivities have been administered to patientswith advanced melanoma (Rousenberg, S. A., et al. (1988), N Engl J Med319:1076-1680; Rosenberg S. A., J. Clin. Oncol. 10:180-199) and althoughsporadic vitiligo has been seen in these patients, no adverseophthalmologic effects related to the possible expression of thesemelanocyte antigens on retinal cells has been observed.

Because HLA-A2 is present in about 50% of individuals and the HLA-A2restricted MART-1 antigen also appears to be widely expressed onmelanomas, immunization with the MART-1 antigen may be particularlyuseful for the development of active immunotherapies.

EXAMPLE 2 Characterization of Immunogenic Epitopes of MART-1

Generation of Melanoma Specific CTL Lines and a Clone from TIL

Melanoma specific CTL lines were generated by culturing a single cellsuspension made from metastatic melanoma with 6000 U/ml of IL2(Cetus-Oncology Division, Chiron Corp. Emeryville, Calif.) as previouslyreported (Kawakami, Y. et al., (1988) J. Exp. Med. 168:2183) A T-cellclone, A42 was established by limiting dilution methods from patient,501.

Assessment of antigen recognition by CTL ⁵¹Cr release cytotoxicityassays and cytokine release assays using ELISA to measure IFN-γ, GM-CSFand TNF-α were performed to analyze the reactivity of TIL as describedin Kawakami, Y. et al. (1988), J. Exp. Med. 168:218 (see Example 1).Melanoma cell lines were established in the laboratory. For analysis ofthe recognition of known antigens by TIL, the COS7 cell line transfectedwith cDNAs encoding either, MART-1, gp100, or tyrosinase related protein(gp75) (Cohen, T. et al., (1990) Nucleic Acids Research 18:2807) alongwith HLA-A2.1 cDNA were incubated with TIL for 20 h and the amount ofIFN-γ secreted into the supernatant was measured by ELISA as describedin Example 1. The cDNA encoding MART-1 (see Example 1) or gp100 (seeExample 3) in plasmid pcDNA3 (Invitrogen, San Diego, Calif.) was clonedfrom a 501mel melanoma cDNA library by screening with TIL1235 orTIL1200, respectively (see Example 1). The cDNA encoding tyrosinaserelated protein(gp75) in pCEV27 plasmid was isolated from 501melmelanoma cDNA library using a probe generated by PCR based on thereported sequence of gp75 (Cohen et al. (1980) Nucleic Acids Research18:2807).

Peptide Synthesis and Identification of Antigenic Peptides

Peptides were synthesized by a solid phase method using a Gilson AMS 422multiple peptide synthesizer. The peptides were purified by HPLC on aVydac C-4 column with 0.05% TFA/water-acetonitrile. To identify theantigenic peptides, TIL lysis of T2 cell lines preincubated for 2h witheach peptide was measured using a ⁵¹Cr release cytotoxicity assay.

HA-A2 Restricted Melanoma Specific TIL

HLA-A2 restricted melanoma specific CTL lines and a clone, A42, wereestablished from lymphocytes infiltrating into tumors of 10 melanomapatients. These TIL recognized autologous and most. allogeneic fresh orcultured melanoma cells expressing HLA-A2, but did not recognize HLA-A2melanomas or HLA-A2⁺ non-melanoma cell lines (Kawakami et al. (1992) J.Immunol 148:638). They also recognized HLA-A⁺ normal culturedmelanocytes derived from neonatal skin (see Example 1 and Kawakami, Y.et al. (1993), J. Immunotherapy 14:88). Thus, these TIL recognizednon-mutated self-peptides derived from proteins expressed in melanomaand melanocytes in association with HLA-A2.

Recognition of Additional Melanoma Proteins by TIL

To evaluate the frequency of recognition of 4 isolated melanoma proteinsincluding MART-1, a form of gp100 (FIG. 5A; SEQ ID NO: 26, see Example3), and tyrosinase related protein(gp75), the reactivity of TIL to COS7was tested on cell lines transfected with cDNAs encoding these 3proteins with or without the cDNA encoding HLA-A2.1. One of severalexperiments with 9 TIL is shown in Table 4. Eight of the nine HLA-A2restricted melanoma specific TIL secreted IFN-γ when incubated with COS7cotransfected with MART-1 and HLA-A2.1. Only TIL1200 which is arelatively oligoclonal CTL line (Shilyansky, J. et al., (1994) Proc.Natl. Acad. Sci. (USA) 91:2829) did not respond to this COStransfectant. Four TIL (620, 660, 1143, 1200) recognized gp100 whentransfected along with HLA-A2.1. TIL1200 secreted large amounts of IFN-γcompared to TIL620, 660, and 1143, suggesting that only a small subsetof T-cells in these latter 3 TIL lines recognized gp100. None of theseTIL recognized gp75 using this assay. Thus, MART-1 is a common melanomaantigen recognized by most HLA-A2 restricted TIL derived from melanomapatients.

Identification of MART-1 Epitopes for TIL

To identify the MART-1 epitopes for these TIL, 23 peptides were selectedbased on the known peptide binding motifs to HLA-A2.1 (Falk, K. et al.,(1993) Nature, 351:290; Hunt, D. F. et al. (1992), Science, 255:1261;Ruppert, J. et al., (1993) Cell 74:929), synthesized (>90% purity) andscreened by testing lysis of the HLA-A2.1⁺ T2 cell line by TIL afterincubation of the T2 line with each peptide (Table 5). The T2 cells(Cerundolo, V. et al.;

TABLE 4 Recognition of melanoma antigens by HLA-A2 restricted melanomaspecific TIL TIL TIL TIL TIL TIL TIL TIL TIL TIL Stimulator Transfected501 620 660 1074 1088 1128 1143 1200 1235 Cell line cDNA HLA-A2 IFN-γsecretion (pg/ml) 501mel none + 93 720 530 670 491 272 354 736 750397mel none − 0 0 0 0 0 0 0 0 0 COS7 none − 0 0 0 0 0 0 14 0 0 COS7HLA-A2.1 + 0 0 0 0 0 0 0 0 0 COS7 MART-1 − 0 0 0 0 0 0 12 0 0 COS7 gp100− 0 0 0 0 0 0 14 0 0 COS7 gp75 − 0 0 0 0 0 0 0 0 0 COS7 HLA-A2.1 +MART-1 + 270 196 131 625 328 52 184 0 743 COS7 HLA-A2.1 + gp100 + 0 8917 0 0 0 41 391 8 COS7 HLA-A2.1 + gp75 + 0 0 0 11 0 0 7 0 0 IFN-γ wasmeasured in the supernatants after HLA-A2 restricted melanoma specificTIL were coincubated with COS7 cells cotransfected with cDNAs encodingproteins expressed in melanoma with or without HLA-A2.1 cDNA. All TILexcept TIL1200 secreted IFN-γ when cultured with COS7 cotransfected withcDNAs encoding MART-1 and HLA-A2.1. TIL620, 660, 1143 and 1200 secretedIFN-γ when cultured with COS7 cotransfected with the cDNAs encodinggp100 and HLA-A2.1.(1990) Nature 345: 449-452) cell line was lysed well by all 4 HLA-A2restricted melanoma specific TIL tested when preincubated with eitherpeptides M9-2, M10-3, or M10-4. Both 10 amino acid peptides, M10-3 andM10-4 contain the M9-2 sequence, with M10-3 having an additionalglutamic acid at its N-terminus and M10-4 having an extra isoleucine atits C-terminal end. These peptides are located in a hydrophobic putativetransmembrane domain in MART-1. The same lysis was observed when otherHLA-A2⁺ cells incubated with these peptides were used as targetsincluding the K4B (provided by Dr. William Biddson, NIH; Storkus. W etal. (1993) J. of Immunology 151:3719-3727) and 501EBVB Epstein-Barrvirus transformed B cells (Topalian et al. (1989) J. Immunol. 142:3714-3725) or HMY-C1R B cells (Dr. William Biddson; NIH; Storkus, W. etal., (1993) J. of Immunol. 151:3719-3727) transfected with the HLA-A2.1gene (data not shown).

The peptides, M9-1, M9-2, M9-3, M10-2, M10-3, M10-4 and M10-5 werefurther purified and titrated in order to evaluate their relativeability to sensitize T2 cells to lysis by MART-1 reactive TIL1235 or Tcell clone A42 (FIG. 2). The purified peptides M9-2, M10-3 and M10-4were required in minimum concentrations of 1 ng/ml, 10 ng/ml an 1000ng/ml, respectively. The purified M10-4 was not recognized by TIL cloneA42 even at 10 ug/ml as shown in FIG. 2. M9-1, M9-3, M10-2, and M10-5peptides were not recognized by either A42 or TIL1235.

TABLE 5 Lysis of T2 cells preincubated with synthetic MART-1 peptidesTIL TIL TIL A42 1235 660 1074 Target Peptide % specific lysis 501melnone 47 30 31 41 397mel none 1 0 1 2 T2 none −2 −3 −1 1 T2 M9-1TTAEEAAGI −10 −5 −5 −4 T2 M9-2 AAGIGILTV 64 80 40 56 T2 M9-3 GIGILTVIL18 20 0 10 T2 M9-4 GILTVILGV 1 −1 −3 2 T2 M9-5 ILTVILGVL −2 −1 −5 −1 T2M9-6 LTVILGVLL 1 0 1 0 T2 M9-7 TVILGVLLL −2 −3 −2 1 T2 M9-8 VILGVLLLI 15 −2 −2 T2 M9-9 ALMDKSLHV −1 −4 −8 0 T2 M9-10 SLHVGTQCA −1 1 −8 4 T2M9-11 PVVPNAPPA −2 0 4 −1 T2 M9-12 NAPPAYEKL 1 −2 0 6 T2 M10-1YTTAEEAAGI −4 −2 −3 3 T2 M10-2 TAEEAAGIGI 7 11 12 15 T2 M10-3 EAAGIGILTV55 66 31 51 T2 M10-4 AAGIGILTVI 34 68 29 21 T2 M10-5 GILTVILGVL −1 2 710 T2 M10-6 ILTVILGVLL 1 6 6 7 T2 M10-7 LTVILGVLLL −2 −1 −1 2 T2 M10-8TVILGVLLLI −6 −1 −1 11 T2 M10-9 RALMDKSLHV 3 5 8 11 T2 M10-10 SLHVGTQCAL−2 −8 2 9 T2 M10-11 SLQEKNCEPV 3 2 2 9 Twenty-three peptides (12 9-mersand 11 10-mers) (>90% purity) were synthesized and the lysability by TILclone A42, TIL lines TIL1235, TIL660, and TIL1074 derived from differentpatients was tested against HLA-A2⁺ T2 cells preincubated with eachpeptide (10 ug/ml) in a 4h-⁵¹ Cr release cytotoxicity assay at E:T ratioof 20:1 for A42 and 40:1 for other TIL lines. T2 cells were lysed wellwhen incubated with M9-2, M10-3 and M10-4. M10-3 and M10-4 contain theentire M9-2 sequence (underlined).

Recognition of MART-1 Peptides by HLA-A2 Restricted TIL Established fromDifferent Patients

To evaluate whether a variety of HLA-A2 restricted MART-1 specific TILrecognized the same or different epitopes in the MART-1 antigen, lysisof T2 cells (Cerundolo V., et al. (1990) Nature 345: 449-452)preincubated with each peptide was tested with TIL derived from 10melanoma patients. A representative experiment with 10 TIL is shown inTable 6. M9-2 and M10-3 were recognized by 9 of 10 TIL (only TIL1200were negative).as well as the A42 clone with the same pattern of lysisas COS7 cells cotransfected with cDNAs encoding MART-1 and HLA-A2.1.Only TIL620 and TIL1088 demonstrated low level of non-specific lysis ofT2 cells without peptides or after the addition of irrelevant peptides,but showed significant increase of lysis of T2 cells preincubated withM9-2, M10-3, and M10-5 peptides. The recognition of M10-4 differed amongthe TIL, but was similar to the different reactivity to M10-4 by theT-cell clone A42 or the T-cell line TIL1235 (FIG. 2). Higherconcentrations (1 ug/ml) of M10-4 were required for lysis than wererequired for M9-2 or M10-3. These 10 TIL and clone A42 also secretedcytokines including IFN-γ, GM-CSF and TNF-α when incubated with T2 cellspreincubated with M9-2 or M10-3 (data not shown). Therefore, M9-2 orM10-3 are common epitopes recognized by a majority of HLA-A2 restrictedmelanoma specific TIL.

TABLE 6 Recognition of MART-1 peptides by HLA-A2 restricted melanomaspecific TIL TIL TIL TIL TIL TIL TIL TIL TIL TIL TIL clone Peptide 501620 660 1074 1088 1128 1143 1200 1235 1363 A42 Target (ug/ml) % specificlysis 501mel none 42 49 35 32 31 19 24 41 32 43 41 397mel none 3 16 6 11 4 3 3 3 4 1 T2 none 0 7 −3 −6 7 −6 −7 −6 −7 −7 −6 T2 M9-1 (1) 4 15 −41 31 1 −5 −1 1 4 3 T2 M9-2 (1) 86 75 73 79 98 30 36 2 92 82 91 T2 M9-2(0.001) 52 49 23 32 81 9 6 1 10 41 63 T2 M9-3 (1) 5 25 0 1 19 0 1 −2 0−2 −4 T2 M10-2 (1) 10 22 5 8 21 8 3 7 7 7 6 T2 M10-3 (1) 84 68 68 73 7924 27 1 42 67 62 T2 M10-3 (0.001) 91 50 33 25 86 13 14 0 14 39 1 T2M10-4 (1) 83 47 16 35 80 6 3 1 14 53 0 T2 M10-4 (0.001) 0 11 3 0 14 4 −1−1 2 −3 −3 T2 M10-5 (1) 4 14 1 4 13 2 3 0 3 0 2 Lysability by TIL cloneA42 and TIL lines derived from 10 patients of T2 cells preincubated withthe purified peptides M9-1, M9-2, M9-3, M10-2, M10-3, M10-4, and M10-5was tested in a 4 h-⁵¹Cr release assay at an E:T ratio of 20:1 for A42and 40:1 for other TIL lines. Nine of ten TIL lysed T2 cells incubatedwith peptides M9-2 or M10-3. Seven of ten TIL lysed T2 incubated withpeptide M10-4 at a concentration of 1 ug/ml.

The relative frequency of recognition of known melanoma proteins byT-cells derived from the TIL of ten melanoma patients has been examined.The common epitopes, M9-2 and M10-3 in the MART-1 antigen that weredominantly recognized by nine of these TIL have also been identified.The cDNA encoding MART-1 was isolated by cDNA expression cloning usingTIL1235 in screening assays (See Example 1). MART-1 is a 118 amino acidprotein containing a single transmembrane domain and is expressed inmost melanoma cells as well as cultured melanocytes and retina similar:to the expression pattern of the cDNA for a form of gp100 described inExample 3. The gp100 is recognized by 4 of 10 TIL.

Based on dose response analysis, peptide M9-2 most effectivelysensitized T2 cells for lysis (FIG. 2) suggesting that this peptide maybe naturally processed and presented on tumor cells. The T-cellsrecognizing M9-2 may react with peptide M10-3 or M10-4 because thelatter 10-mer peptides contain the 9 amino acid sequence of peptideM9-2. There is some difference in recognition of these 3 peptides bydifferent TIL. For example, M10-4 was poorly recognized by the T-cellclone A42, but was well recognized by some TIL lines, although a higherconcentration of M10-4 was necessary to observe the lysis. This may bedue to the variation of TCR affinity for the M9-2 and M10-4 peptides inthe context of HLA-A2, or alternatively, TIL lines may contain differentT-cell clones which only recognize either M9-2 or M10-4. Peptides M10-3and M10-4 may also be naturally processed and presented by tumor cells.The existence of multiple melanoma antigens presented by HLA-A2 haspreviously been suggested by analyzing the recognition of melanoma cellclones by a variety of T-cell clones (Knuth, A. et al. (1989), Proc.Natl. Acad. Sci. (USA) 86:2804, Wolfel, T. et al., 1989 J. Exp. Med.170:797) or by analyzing HPLC peptide fractions that were isolated fromHLA-A2 melanoma cells (Slingluff, C. L. Jr. et al., (1993) J. Immunol.150:2955, Storkus; W. J. et al., (1993) J. Immunol. 151:3719).

The observation that most HLA-A2 restricted TIL from melanoma patientsrecognize common MART-1 peptides but not gp75 suggests that the M9-2 orM10-3 MART-1 peptides may be more immunogenic in inducing T-cellresponses in vivo than other known melanoma antigens. Some of the TILused in this study were injected along with IL2 into autologouspatients, and interestingly, all 4 TIL (620, 660, 1074, 1200) thatrecognize a gp100 protein (FIG. 5A; SEQ ID NO: 27) effectively inducedtumor regression (more than 50% reduction of tumor). All but TIL1200also recognized MART-1.

EXAMPLE 3 Identification of a Second Human Melanoma Antigen Recognizedby Tumor Infiltrating Lymphocytes Associated with in Vivo TumorRejection cDNA Expression Cloning

The cDNA25 clone encoding a form of the melanoma antigen designatedgp100 was cloned by techniques similar to those described in Example 1and in Miki, T., et al. (1991) Proc. Natl. Acad. Sci. (USA)88:5167-5171. Briefly, a breast cancer cell line, MDA231 (ATCC #HTB26),transfected with a cDNA library in λpCEV27 made from the 501mel melanomacell line was screened for antigen positivity by measuring interferon-λ(IFN-γ) secretion when cocultured with TIL1200. TIL1200 was generated asdescribed in Kawakami, Y., (1988), J. Exp. Med. 168, 2183-2191. Theintegrated cDNA was recovered from the genomic DNA of positivetransfectants by PCR and cloned into the mammalian expression plasmidpcDNA3 (Invitrogen, San Diego, Calif.). The full length cDNA for cDNA25was isolated from the 501mel λpCEV27 library using the cDNA25 probe. Theλphage containing the full length cDNA25 was digested with XhoI, andthen self-ligated with T4 DNA ligase to make the plasmid pCEV27-FL25.Alternatively, a full length cDNA25 isolated by PCR using the specificprimers designed for gp100 was cloned in pCRII(Invitrogen), and thencloned into pcDNA3(pcDNA3-FL25). To test whether this cDNA encoded amelanoma antigen it was retransfected into COS7, A375 or MDA231 and theresulting transfectants were tested for stimulation of TIL1200. DNAsequences of the plasmid clones were determined with an automated DNAsequencer (Model 373A; Applied Biosystems, Inc.), using the Taq DyeDeoxyterminator cycle sequencing kit (Applied Biosystems, Inc.) using themanufacturer's instructions.

Peptide Synthesis and Identification of Antigenic Peptides

Peptides were synthesized by a solid phase method using a Gilson AMS 422multiple peptide synthesizer. The peptides were purified by HPLC on aVydac C-4 column with 0.05% TFA/water-acetonitrile. To identifyantigenic peptides, TIL lysis of T2 RET-cells preincubated with peptidesfor 2 hour (h) was measured using a ⁵¹Cr release cytotoxicity assay.

Treatment of a Patient with Metastatic Melanoma Using TIL 1200

A 29 year old male patient, designated patient number 1200, with awidely metastatic melanoma who had previously failed chemotherapy andradiation therapy was treated with a single preparatory dose of 25 mg/Kgcyclophosphamide followed by the intravenous infusion of 1.6×10¹¹ TIL(including 9.1×10⁹ Indium-ill labeled TIL) plus 7 doses of IL-2 at720,000 IU/Kg given every 8 hours. A second cycle of treatment with TILand IL-2 was given three weeks later. Radionuclide scans showedlocalization of TIL in rumor deposits (FIG. 3A). Biopsy of subcutaneoustumors on days 8 and 11 after treatment showed significant localizationof TIL to tumor (ratios of injectate per gram in tumor compared tonormal tissue were 14.9 and 14.0 respectively). The patient's cancerregressed rapidly following the first course of treatment. By threemonths after treatment 2 of 3 liver lesions had disappeared and a thirdlesion shrank by 50%. Multiple subcutaneous metastases regressedcompletely as shown in FIG. 3B (the product of perpendicular diametersof individual lesions are shown).

Characterization of in vitro function of TIL1200 A number of TIL linesestablished from HLA-A2⁺ melanoma patients lysed melanoma cell lines ina class I MHC-restricted fashion (Kawakami, Y., et al. (1992) J.Immunol. 148; 638-643), and were shown to release IFNγ, tumor necrosisfactor-alpha (TNFα) or granulocyte-macrophage colony stimulating factor(GM-CSF) when cocultured with the same tumor cell lines (Hom, S-S., etal. (1993) J. Immunother. 13; 18-30). A CD8⁺ CTL line, TIL1200,established from a metastatic subcutaneous tumor mass of patient 1200,lysed fresh autologous melanoma cells as well as 10 of 15 HLA-A2⁺allogeneic melanoma cell lines, but did not lyse 16 of 18 HLA-A2⁻melanoma cell lines or 6 of 8 HLA-A2⁺ non-melanoma cell lines(Shilyansky, J., et al. (1993) Proc. Natl. Acad. Sci. USA, 91,2829-2833, unpublished data). Table 7 shows a cytotoxicity assay against5 representative HLA-A2⁺ melanoma cell lines that were lysed by TIL1200,4 representative HLA-A2+ melanoma cell lines that were not lysed byTIL1200, and one HLA-A2⁻ melanoma cell line. TIL1200 also secreted IFN-γwhen cocultured with HLA-A2⁺ normal cultured melanocytes establishedfrom neonatal foreskin as well as HLA-A2⁺ melanoma cell lines (Table 8).Therefore, TIL1200 appeared to recognize a non-mutated self peptideexpressed in most melanomas and cultured neonatal melanocytes in anHLA-A2 restricted fashion.

Cloning of the cDNA Coding for a Melanoma Antigen Recognized by T Cells

A cDNA library in λ pCEV27 from the HLA-A2⁺ 501mel melanoma cell line,which was lysed by most HLA-A2 restricted melanoma specific TIL, wasstably transfected into the highly transfectable HLA-A2⁺ melanomaantigen negative MDA231 clone 7 or A375 clone 1-4. G418 resistant cellswere selected and approximately 6700 individual transfectants from eachcell line were isolated and screened based on their ability to stimulateIFN-γ secretion from TIL1200. Six DNA fragments were isolated by PCRusing SP6/T7 primers flanking the integrated DNA from four MDA231 andone A375 transfectants that were positive in a second screening and werecloned into the mammalian expression vector pcDNA3 (Invitrogen).

These fragments in the pcDNA3 vector were transiently expressed in theCOS7 cells with or without pcDNA3-HLA-A2.1. Transfection into COS7 ofone of the cDNAs tested, cDNA25, along with HLA-A2.1 reproduciblyconferred the ability to stimulate secretion of IFN-γ from TIL1200. Thestable transfection of cDNA25 into A375 also stimulated IFN-γ releasefrom TIL1200 (Table 9, Exp.1 and Exp. 2). A 2.2 Kb band detected byNorthern blot analysis of the melanoma using the cDNA25 probe suggestedthat the cloned 1.6 Kb fragment was not a full length cDNA. Comparisonwith the GenBank database of the consensus DNA sequence of

TABLE 7 Specificity of Antigen Recognition by TIL1200: Lysis ofHLA-A2⁺gp100⁺ Melanoma Cell Lines TIL1200 LAK gp100 (% specific TargetHLA-A2 FACS Northern lysis) 501mel + + + 46 78 526mel + + + 39 74624mel + + + 33 76 952mel + + + 25 76 Malme3M + + + 43 70 C32 + − −/+* 682 RPMI7951 + − − 9 67 WM115 + − − 5 68 HS695T + − − 3 87 397mel − + + 070 5 hour (h) ⁵¹Cr release assay was performed to measure cellularcytotoxicity at an effector: target ratio of 40:1 as previouslydescribed(Kawakami, Y. et al. (1988) J. Exp. Med. 168: 2183-2191). Theexpression of HLA-A2 and gp100 recognized by monoclonal antibody HMB45(Enzo Diagnostics, New York, NY) was measured by flow cytometry(FACS).The expression of gp100 RNA was analyzed by Northern blot with a cDNA25probe. *−/+ indicates very weak positive.

TABLE 8 Specificity of Antigen Recognition by TIL1200: Recognition ofHLA-a-a2⁺ Neonatal Melanocytes TIL1200 TIL888 Stimulator HLA-A2(pgIFN-γ/ml) 501mel + 562 0 624mel + 439 0 397mel − 0 0 888mel − 0 1970NHEM493 − 441 0 NHEM527 + 418 0 NHEM530 + 164 0 NHEM616 + 53 0 FM725 +107 0 FM801 + 250 343 NHEM483 − 0 0 NHEM680 − 0 0 HA002 − 0 0 The IFN-γsecretion by TIL was measured by ELISA as previously described inExample 1. The amount of IFN-γ secreted by TIL alone was subtracted (88pg/ml for TIL888 and none for TIL 1200). TIL888 is a class I MHCrestricted melanoma specific CTL, not restricted by HLA-A2. NHEM, FM,and HA refer to normal cultured melanocyte cell lines, all others aremelanoma cell lines.

TABLE 9 Transfection of cDNA 25 into A375 and COS7 Secretion byStimulator Transfected HLA- TIL1200 cells genes A2 (pgIFNγ/ml) Exp. 1501mel none + 987 397mel none − 0 A375 none + 0 A375 pcDNA3-25 + 230Exp. 2 501mel none + 662 397mel none − 0 COS7 none − 0 COS7 HLA-A2.1 + 0COS7 pcDNA3-25 − 0 COS7 HLA-A2.1+pcDNA3-25 + 310 Exp. 3 501mel none +908 397mel none − 0 COS7 none − 0 COS7 HLA-A2.1 + 0 COS7 pCEV27-FL25 − 0COS7 HLA-A2.1+pCEV27-FL25 + 742 COS7 pcDNA3-FL25 − 0 COS7HLA-A2.1+pcDNA3-FL25 + 801 TIL 1200 secreted IFN-γ when coincubated withHLA-A2⁺ A375 stably transfected with pcDNA3 containing truncated cDNA25(pcDNA3-25) (Exp. 1) or COS7 transiently transfected with eitherpcDNA3-25 (Exp. 2), pcDNA3 containing full length cDNA25 (pcDNA3-FL25)or pCEV27 containing full length cDNA25 (pCEV27-FL25) (Exp. 3) alongwith pcDNA3 containing HLA-A2.1 (HLA-A2.1). HLA-A2 expression wasdetermined by flow cytometry and interferon-gamma secretion was measuredby ELISA.3 cDN25 clones that were independently amplified by PCR revealed thatcDNA25 was distinct from two previously registered genes, a gp100(GenBank Access No. M77348) and Pmel17 (Kwon, B. S., et al. (1991) Proc.Natl. Acad. Sci, USA 88, 9228-9232). The cDNA 25 differed from the gp100in GenBank (Accession No. M77348, also known as gp95) by twonucleotides, from the PMEL 17 sequence (Kwon et al. (1991) Proc. Natl.Acad. Sciences (USA) 58: 9228-9232) by 3 bases and a 21 base pairdeletion. (FIG. 5B).

The full length cDNA25(FL25) was isolated in two plasmids, pCEV27-FL25or pcDNA3-FL25. Transfection of either plasmid into COS7 along withpcDNA3-HLA-A2.1 conferred to COS7 the ability to induce IFN-γ secretionby TIL1200. The amount of IFN-γ secretion stimulated by COS7 transfectedwith the full length DNA plus HLA-A2.1 was similar to that stimulated by501mel and was higher than that stimulated by COS7 transfected with thetruncated cDNA25 possibly due to improved translation starting at thenormal AUG initiation codon (Table 9, Exp.2 and 3). Alternatively, the5′ region missing from the truncated cDNA25 may contain other epitopesrecognized by clones in TIL1200. The requirement for HLA-A2.1 expressionfor IFN-γ release from TIL1200 and the fact that transfected cells didnot stimulate IFN-γ secretion from irrelevant TIL (data not shown)demonstrated that the cDNA25 encoded an antigen recognized by TIL1200 inthe context of HLA-A2.1 and did not encode a molecule thatnon-specifically induced IFN-γ release from T cells.

The nucleotide and corresponding amino acid sequences of the truncatedcDNA 25 and the full length cDNA25 cloned from the 501mel cDNA libraryby screening with the cDNA25 probe (FIG. 5A) were compared with theGenBank sequences of Pmel17 isolated from normal melanocytes and gp100isolated from the melanoma cell line MEL-1. (FIG. 5B). The full lengthcDNA25 differed from the gp100 amino acid sequence at position 162. Thisamino acid difference is possibly caused by polymorphism or mutation inthe tumor. cDNA25 had 2 amino acid differences at positions 162 and 274,compared to Pmel17 and did not contain 7 amino acids that existed inPmel17 at positions 588-594. The amino acid sequence of the truncatedcDNA25 that was isolated from the original MDA231 transfectant has adifferent sequence at the 3′ end (from position 649 to the end) due to aframe shift caused by one extra cytidylic acid. It is not clear whetherthis difference was due to a true allelic difference or to a mutationthat occurred during manipulation of the DNA. Nevertheless, TIL1200appeared to recognize non-mutated peptides located between position 236and 648. cDNA25 also had 87% similarity in amino acid sequence to cDNARPE1 (Kim, R., and Wistow, G. J. (1992) Exp. Eve Res. 55: 657-662)specifically expressed in bovine retinal pigment epithelium and 60%similarity to cDNA MMP115 that encoded a melanosomal matrix proteinisolated from chicken pigmented epithelial cells (Shilyansky, J., et al.(1993) Proc. Natl. Acad. Sci, USA, 91, 2829-2833).

A gp100 protein was known to be recognized by monoclonal antibody HMB45(Adema et al., (1993) Am. J. Patholoay, 143: 1579-1585). COS7 cellstransfected with the full length cDNA25 were evaluated by flow cytometryusing this monoclonal antibody. After transient expression of eitherpCEV27-FL25 or pcDNA3-FL25, COS7 expressed the antigen detected by HMB45(data not shown).

Expression of RNA for cDNA25

Northern blot analysis was performed with the cDNA25 probe to evaluatethe tissue specific expression of this gene. Ten of 15 melanoma celllines and 6 of 6 melanocyte cell lines were positive for cDNA 25. Ofmany normal tissues tested only retina was positive (FIG. 6). Seven celllines from T-cell (TILA, B), B-cells (501EBVB, 836EBVB) and fibroblast(M1) and 20 non-melanoma tumor cell lines (colon cancer, Collo, SW480,WiDr; breast cancer, MDA231, MCF7, HS578, ZR75; neuroblastoma, SK—N-AS,SK—N—SH; Ewing sarcoma, TC75, RD-ES, 6647; sarcoma 143B; glioma, U138MG,HS683; renal cell cancer, UOK108, UOK117, small cell lung cancer, H1092;Burkitt's lymphoma, Daudi; myeloma HMY) were all negative for cDNA25(data not shown). Therefore, this gene appeared to be specificallyexpressed in melanocyte lineage cells, consistent with the expressionpattern of previously isolated forms of gp100 when analyzed usingmonoclonal antibodies, HMB45, NKI/betab, or HMB-50 (Adema, G. J., et al.(1993) Am J Pathology 143: 1579-1585; Gown, A. M., et al., (1986) Am JPathol 123:195-203; Colombari, R., et al. (1988) Virchows Archiv APathol Anat. 413:17-24); Vennegoor, C., et al. (1988) Am. J. Pathol.130:179-192; Vogel, A. M., and Esclamado R. M. (1988) Cancer Res.48:1286-1294). The levels of expression of the RNA detected by thecDNA25 probe in cultured neonatal melanocyte cell lines wassignificantly lower than that in melanoma cell lines. There was aperfect correlation between gp100 expression detected by Northern blotanalysis with cDNA25 and flow cytometry using HMB45 antibody andmelanoma lysis by TIL1200 in the 10 HLA-A2⁺ melanoma cell lines as shownin Table 7.

Identification of the Epitope in gp100

Based on a comparison of the amino acid sequence of the truncated formof cDNA 25 to known binding motifs of HLA-A2.1 (Falk, K., et al. (1992)Nature 351:290-296; Hunt, D. F., et al. (1992) Science 255:1261-1263;Ruppert, J., et al. (1993) Cell 74:929-9937,) 30 peptides of 9 or 10amino acids in length from cDNA25 were synthesized. TIL1200 lysed theHLA-A2⁺ cell line, T2, only when incubated with the peptide LLDGTATLRL(SEQ ID NO: 27 residues 457-486. FIG. 5A; SEQ ID NO: 33) but not whenincubated with the other 29 peptides (Table 10, FIG. 5A). Only peptideLLDGTATLRL (SEQ ID NO: 33) was able to also stimulate IFN-γ secretion byTIL 1200 (data not shown).

Many melanoma-specific CTL derived from TIL appear to recognizenon-mutated self peptides derived from melanocyte-melanoma lineagespecific proteins, since these TIL recognize most melanoma cell linesand normal cultured melanocytes sharing the appropriate restrictionelement. (Anichini, A., et al. (1993) J. Exp. Med. 177:989-998;Kawakami, Y., et al. (1993) J. Immunother. 14:88-93). In an attempt toisolate and identify melanoma antigens of value in the immunotherapy ofmelanoma patients, TIL, TIL1200 were used that, when transferred into apatient with metastatic cancer, localized to the tumor site and wasassociated with a dramatic tumor regression. It has been shown that, incontrast to non-activated lymphocytes and lymphokine activated killercells, autologous TIL localize to tumor sites. This localizationcorrelated with the ability of these TIL to mediate tumor regression(data not shown). TIL1200 which was a TIL line containing multiple CTLspecies recognized a tumor antigen in the context of HLA-A2, which isthe most frequently expressed class I MHC antigen (about 50% ofindividuals) and has been shown to be a dominant restriction element forthe induction of melanoma specific CTL. (Crowley, N. J., et al. (1991)J. Immunol. 146, 1692-1699).

By cDNA expression cloning using T cell recognition for screening, acDNA (FIG. 4; SEQ ID NO: 26) encoding an antigen recognized by TIL1200and identified as a form of gp100, a membrane glycoprotein alsorecognized by monoclonal antibodies, BMB45, HMB50 or NKI/betab has beenidentified. (Adema, G. J., et al. (1993) Am J Pathology 143, 1579-1585.Gown, A. M., et al. (1986) Am J Pathol 123, 195-203. Colombari, R., etal. (1988) Virchows Archiv A Pathol Anat. 413, 17-24; Vennegoor, C., etal. (1988) Am. J. Pathol. 130, 179-192; Vogel, A. M., and Esclamado R.M. (1988) Cancer Res. 48, 1286-1294). These antibodies are highlyspecific for melanocyte lineage tissues and strongly stain most melanomacells. NKI/betab also reacts with adult melanocytes in normal skin(Vennegoor, C., et al. (1988)

TABLE 10 TIL1200 Lysis of the T2 HLA-A2⁺ Cell Line Pulsed with thePeptide, LLDGTATLRL Peptide* TIL1200 TIL1235⁺ Target HLA-A2 (ug/ml) (%specific lysis)** 501mel + 0 66 51  397mel − 0 1 0 T2 + 0 2 1 T2 + 40 28ND⁺⁺ T2 + 10 32 0 T2 + 1 24 ND T2 + 0.1 6 ND T2 + 0.01 0 ND T2 + 0.001 2ND *TIL1200 lysed T2 cells pulsed with the 10-mer peptide,LLDGTATLRL(457-466), but not other 29 peptides SEQ ID NO: 27 (residues273-281, 297-306, 373-381, 399-407, 399-408, 409-418, 456-464, 463-471,465-473, 476-485, 511-520, 519-528, 544-552, 544-553, 570-579, 576-584,576-585, 585-593, 592-600, 597-605, 597-606, 602-610, 602-611, 603-611,605-614, 606-614, 606-615, 619-627, 629-638) ⁺TIL1235 is an HLA-A2restricted melanoma specific CTL that does not recognize gp100. **E:T of50:1 ⁺⁺ND, not done.Am. J. Pathol. 130, 179-192) Immunoelectron-microscopic studies usingeither HMB45 or NKI/betab antibody revealed that a gp100 protein wasmainly located in a membrane and filamentous matrix of stage I and IImelanosomes in the cytoplasm (Vennegoor, C., et al. (1988) Am. J.Pathol. 130, 179-192; Schaumburg-Lever, G., et al. (1991) J. Cutan.Pathol. 18, 432-435). By a completely independent procedure, a cDNAencoding another form of gp100 was also isolated by screening with arabbit polyclonal antiserum against gp100 (Adema, G. J., et al. (1993)Am J Pathology 143:1579-1585) and TIL1200 also lysed HLA-A2⁺ cell linestransfected with this cDNA clone (Bakker, A. B. H. et al. (1994) J. Exp.Med. 179:1005-1009).

The existence of T cells reactive to the self-antigen gp100 in tumorsand the possible enrichment of these T cells at the tumor site as apossible consequence of the specific accumulation and expansion ofantigen reactive cells (Sensi, M. et al., (1993) J. Exp. Med178:1231-1246) raises important questions about the nature of the immuneresponse to self antigens on growing cancers and about the mechanisms ofimmunologic tolerance to self-antigens. The increased expression ofgp100 on melanoma cells relative to that in melanocytes demonstrated byNorthern blot analysis or the unique inflammatory conditions that mightexist at the tumor site, which may be associated with the secretion ofcytokines and expression of costimulatory molecules on the cell surface,could break tolerance to gp100. Depigmentation has been reported to beassociated with a good prognosis (Nordlund, J. J., et al. (1983) J. Am.Acad. Dermatol. 9:689-695; Bystryn, J-C, et al. (1987) Arch. Dermatol.,123:1053-1055) and with clinical response to chemoimmunotherapy(Richards, J. M., et al. (1992) J. Clin. Oncol. 10:1338-1343) inmelanoma patients. Sporadic vitiligo in has been seen patients receivingmelanoma-specific TIL but adverse ophthalmologic effects that might berelated to melanocyte destruction has not been observed. Patient 1200did not develop vitiligo or any ophthalmologic side-effects.

The gp100 protein (FIG. 5A; SEQ ID NO: 27) and the ten amino acidpeptide identified may represent a human tumor rejection antigen sincethe transfer into patient 1200 of TIL1200 plus IL2 was associated withcancer regression. The traffic of TIL1200 to tumor deposits in vivo andthe rapidity of the antitumor response are characteristics of theresponse to TIL therapy, although IL2 may also have been involved in thetumor rejection. Adoptive transfer of 3 other TIL lines which recognizedgp100 as well as MART-1 also mediated tumor regression (data not shown).

Tyrosinase (Brichard, V., et al. (1993) J. Exp. Med. 178, 489-495) andMART-1 (see Example 1) have been identified as melanoma antigensrecognized by HLA-A2 restricted CTL. Another antigen, MAGE-1 isrecognized by HLA-A1 restricted melanoma-specific CTL and is expressedon a variety of cancer cells as well as testis (Van Der Bruggen, P. etal. (1991) Science, 254:1643-1647). However, none of the ten HLA-A2restricted TIL recently developed appeared to recognize MAGE-1 (Zakut,R., et al. (1993) Cancer Res. 53: 5-8).

The wide expression of gp100 proteins in melanomas, the recognition of apeptide by T cells infiltrating into tumor, its restriction by HLA-A2,present in 50% of individuals, and the association of anti gp100reactivity with cancer regression in patient 1200 imply that the gp100antigen in particular the novel immunogenic peptides derived from thegp100 amino acid sequence (FIG. 5A; SEQ ID NO: 27) may be particularlyuseful for the development of active immunotherapies for patients withmelanoma.

EXAMPLE 4 Recognition of Multiple Epitopes in Human Melanoma Antigen byTIL Associated with in Vivo Tumor Recognition Materials and MethodsGeneration of CTL from TIL and Treatment of Patients with MetastaticMelanoma

Melanoma specific CTL were induced and expanded from TIL in mediacontaining 6000 IU/ml of IL2 as previously described (Kawakami, et al.,(1988) J. Exp. Med. 168:2183). All available HLA-A2 restricted melanomaspecific CTL which were administered to autologous patients in theSurgery Branch, NCI, were used in this study. TIL were administeredintravenously along with IL2 into autologous patients with metastaticmelanoma as previously reported (Rosenberg, S. A., et al., (1988) N EnglJ Med 319:1676; Rosenberg S. A., et al., (1994) J. NCI. 86:1159).Fisher's exact test was used to determine the association of gp100recognition by TIL with clinical response to TIL treatment; likewisewith MART-1 recognition.

Synthesis of Peptides

Peptides were synthesized by a solid phase method using a peptidesynthesizer (model AMS 422;Gilson Co.Inc., Worthington, Ohio)(>90%purity). The peptides to be synthesized were selected from the reportedhuman sequence of gp100 based on HLA-A2.1 binding motifs (Falk, K.,(1991) Nature 351:290; Hunt, D. F., et al, (1992) Science 255:1261;Ruppert, J., et al., (1993) Cell 74:929; Kubo, R T, et al. (1994) JImmunol. 152:3913). The following peptides were tested: Eight 8-merpeptides (with residues starting at −199, 212, 218, 237, 266, 267, 268,269; see FIG. 7A), eighty-four 9-mer peptides with residues starting at−2, 4, 11, 18, 154, 162, 169, 171, 178, 199, 205, 209, 216, 241, 248,250, 255, 262, 266, 267, 268, 273, 278, 280, 273, 286, 287, 296, 290,309, 316, 332, 335, 350, 354, 358, 361, 371, 373, 384, 389, 397, 399,400, 402, 407, 408, 420, 423, 425, 446, 449, 450, 456, 463, 465, 485,488, 501, 512, 536, 544, 563, 570, 571, 576, 577, 578, 583, 585, 590,592, 595, 598, 599, 601, 602, 603, 604, 606, 607, 613, 619, 648; seeFIG. 7A) and seventy-seven, 10-mer peptides with residues starting at−9, 17, 57, 87, 96, 154, 161, 169, 177, 197, 199, 200, 208, 216, 224,232, 240, 243, 250, 266, 267, 268, 272, 285, 287, 289, 297, 318, 323,331, 342, 350, 355, 357, 365, 380, 383, 388, 391, 395, 399, 400, 406,407, 409, 415, 432, 449, 453, 457, 462, 476, 484, 489, 492, 511, 519,536, 543, 544, 548, 568, 570, 571, 576, 577, 584, 590, 595, 598, 599,601, 602, 603, 605, 611, 629; see FIG. 7A) were synthesized. Possibleepitopes identified in the first screening were further purified by HPLCon a C-4 column (VYDAC, Hesperia, Calif.)(>98% purity) and the molecularweights of the peptides were verified by mass spectrometry measurementas previously described (Example 3; Kawakami, Y., et al., (1994) J. Exp.Med. 180:347; Kawakami, Y., et al., (1994) Proc Natl Acad Sci (USA)91:6458).

Peptide Binding Assay to HLA-A2.1

Soluble HLA-A2.1 heavy chain, human beta 2-microglobulin, radiolabeledpeptide HBc₁₈₋₂₇ (FLPSDYFPSV) and various concentrations of the samplepeptides were coincubated in the presence of protease inhibitors for 2days at room temperature as previously described (Ruppert, J., et al.,(1993) Cell 74:929; Kubo, R T, et al., (1994). J Immunol. 152:3913; SettA., et al., (1994). Molecular Immunol. 31:813). The percentage oflabeled peptide bound to HLA-A2.1 was calculated after separation by gelfiltration and the concentration of the sample peptide necessary toinhibit 50% of the binding of the labeled peptide was calculated. Therelative affinity of peptides to HLA-A2.1 were also calculated as aratio (concentration of the standard HBc₁₈₋₂₇ peptide to inhibit 50% ofthe binding of the labeled peptide/concentration of the sample peptideto inhibit 50% of the binding of the labeled peptide) as previouslydescribed (Sett A., et al., (1994) Molecular Immunol. 31:813). Peptidebinding was defined as high (50% inhibition at <50 nM, ratio>0.1),intermediate (50-500 nM, ratio 0.1-0.01) or weak (>500 nM, ratio<0.01)(Ruppert, J., et al., (1993) Cell 74:929; Kubo, R T, et al., (1994) JImmunol. 152:3913; Sett A., et al., (1994) Molecular Immunol. 31:813).

The pcDNA3 plasmid containing the full length gp100 cDNA (FIG. 4,Example 3; Kawakami, Y., et al., (1994)). Proc Natl Acad Sci (USA)91:6458) was digested with Xho I and Xba I. After incorporation ofalpha-phosphorothioate deoxynucleoside triphosphate into the Xba I site,a standard exonuclease III nested deletion was performed using the ExoSize Deletion Kit (New England Biolabs, inc., Beverly, Mass.). Thedeleted clones were self-ligated and amplified. The exact deletion foreach clone was confirmed by DNA sequencing. To identify the regioncontaining epitopes, pcDNA3 plasmids (Invitrogen, San Diego Calif.)containing the cDNA fragments (D3, D5, D4, C3) generated by thesequential deletion with exonuclease from the 3′ end of the full lengthgp100 cDNA as well as the truncated gp100 cDNA lacking the 5′-codingregion (25TR) (Example 63; Kawakami, Y. (1994) Proc Natl Acad Sci (USA)91:6458), were transfected into COS7 cells along with the HLA-A2.1 cDNAand the recognition of the transfected COS cells by TIL was evaluatedusing IFN-γ release assays (Example 1; Kawakami, Y., (1994) Proc NatlAcad Sci (USA) 91:3515).

Evaluation of Antigen Recognition by T-Cells

To assess antigen recognition by T-cells, a ⁵¹Cr release assay or anIFN-γ release assay were performed as previously described (Examples 1and 2; Kawakami, Y., et al., (1994)). Proc Natl Acad Sci (USA) 91:3515;Kawakami, Y., et al., (1988). J. Exp. Med. 168:2183). Either COS7 cellstransfected with cDNA encoding melanoma antigens and HLA-A2.1 cDNA, orT2 cells preincubated with peptides were used as stimulators for theIFN-γ release assay. T2 cells pulsed with peptides were also used astargets for cytotoxicity assays (Kawakami, Y., (1994) J. Exp. Med.180:347).

Recognition of gp100 by TIL Correlated with Clinical Response to TILTreatment

Four of 14 HLA-A2 restricted melanoma specific CTL derived from TILrecognized gp100 while 13 recognized MART-1 (3 recognized both gp100 andMART-1). None recognized tyrosinase or gp75 as assessed by thereactivity of TIL against COS7 cells transfected with the cDNA encodingthese melanoma antigens along with HLA-A2.1 cDNA (Example 2; Kawakami, Yet al. (1994) J. Exp. Med. 180:347). The HLA-A2 restriction and therecognition specificity of these 4 gp100 reactive CTL has beenpreviously demonstrated (Examples 1-3; Kawakami, Y., et al., (1994) ProcNatl Acad Sci (USA) 91:6458; Kawakami, Y., et al., (1992) J Immunol148:638; O'Neil, B. H., et al., (1993) J Immunol 1410:1418; Shilyansky,J., et al., (1994) Proc. Natl. Acad. Sci. (USA) 91:2829). Ten of these14 CTL were administered into the autologous patients along with IL2. Assummarized in Table 11, all 4 patients treated with CTL capable ofrecognizing gp100 resulted in an objective partial response (>50% tumorregression). Clinical response to TIL therapy associated with reactivityof TIL to gp100 (p=0.0048) but not to MART-1 (p=0.4). These datasuggested that gp100 may contain epitopes capable of mediating in vivotumor regression.

Identification of Epitopes Recognized by gp100 Reactive TIL

To identify the epitopes recognized by these 4 gp100 reactive CTL, a 169peptides which contained HLA-A2.1 binding motifs were synthesized.Peptide recognition was evaluated by testing the reactivity of these CTLagainst HLA-A2.1+ T2 cells preincubated with each peptide using bothcytotoxicity and IFN-γ release assays. As shown in Table 12, 7 peptideswere recognized by gp100 reactive TIL in the cytotoxicity assays. Theresults of the IFN-7 release assays performed at the same time wereconsistent with that of the cytotoxicity assays. The differentsubcultures of TIL620 (620-1, 620-2) or TIL660 (660-1, 660-2, 660-3)were grown from the TIL culture that was administered into theautologous patient, but they were separately cultured and had slightlydifferent specificities likely due to the in vitro expansion ofdifferent clones. G9₂₀₉ (ITDQVPFSV) (SEQ ID NO:48) and G10₂₀₉(TITDQVPFSV) (SEQ ID NO:49), which has an extra threonine at theN-terminus of G9₂₀₉, were recognized only by TIL620. G9₁₅₄ (KTWGQYWQV)(SEQ ID NO:46) and G10₁₅₄ (KTWGQYWQVL) (SEQ ID NO:47), which has anextra leucine at the C-terminus of G9₁₅₄, were recognized by TIL1200,TIL620-2 and TIL660-2. G10-4 (LLDGTATLRL) (SEQ ID NO:33) was recognizedby TIL1200 as demonstrated (Example 3). The peptide G9₂₈₀ (YLEPGPVTA)(SEQ ID NO:40) was recognized by TIL660 and TIL1143. TIL660-3 alsorecognized G10-5 (VLYRYGSFSV) (SEQ ID NO:34) as well as G9280. Lysis ofT2 cells preincubated with G10-5 was repeatedly low, possibly because asmall subset of T-cell clones was specific for this epitope.

To complement the epitope identification using the known HLA-A2.1binding motifs, another method was also used to identify regionspossibly containing epitopes. Five gp100 cDNA fragments, 4 generated byexonuclease deletion from the 3′-end of the cDNA (D3, D4, D5, C4) aswell as a partial cDNA clone lacking the first 705 base pairs of the5′-coding region (25TR), were inserted into the pcDNA3 plasmid andtransfected into COS7 cells along with the HLA-A2.1 cDNA. The locationsof the fragments are shown in FIG. 7A. The recognition of thesetransfectants by the 4 gp100 reactive TIL was evaluated using an IFN-γrelease assay (FIG. 7B). TIL 1200 recognized COS cells transfected withthe fragments, 25TR, D5, D4 or C4, but not with D3, suggesting that atleast 2 epitopes existed in the regions of amino acid residues 146-163and 236-661. G9₁₅₄ and G10₁₅₄ were the only peptides which containedHLA-A2.1 binding motifs in the region 146-163 and both were recognizedby TIL1200. G10-4 was located in the region 236-661 and was recognizedby TIL1200. TIL620-1 recognized COS cells transfected with C4 but notwith D3, D5, D4 or 25TR, suggesting that the epitope existed withinresidues 187-270. G9₂₀₉, and G₁₀ ₂₀₈ which were recognized by TIL620-1were located in this region. TIL620-2, another subculture of TIL620,also recognized COS cells transfected with D5 and D4, but not D3, andrecognized G9₁₅₄ and G20₁₅₄ in the region 147-163, also recognized byTIL1200. TIL660-1 and TIL1143 recognized COS cells transfected with C4or 25TR, but not with D3, D5, or D4, suggesting that epitopes existed inthe 2 regions 187-270 and 236-661. G9₂₈₀ located in the fragment 25TR,but not in the fragment C4, was recognized by TIL660 and TIL1143.

Binding Affinity of the Melanoma Epitopes to HLA-A2.1 in Vitro

With the exception of G10-4, which required a concentration of 1 ug/mlto sensitize T2 cells for CTL lysis (Example 3; Kawakami, Y., et al.,(1994) Proc Natl Acad Sci (USA) 91:6458), all gp100 epitopes identifiedin this study could sensitize T2 cells for CTL lysis at a concentrationof 1 ng/ml (FIGS. 8A-8D). G10-5 appeared to be inhibitory to thecytotoxic activity of CTL at concentration greater than 10 ng/ml sincelysis of T2 cells incubated with G10-5 at more than 10 ng/ml wasrepeatedly lower than at 1-10 ng/ml in this assay condition in which thepeptide was present in the medium during entire 4 h cytotoxicity assay(FIG. 8D). The relative binding affinity of these epitopes to HLA-A2.1was also measured using an in vitro competitive binding assay (Table13). G9₁₅₄ had an higher binding affinity (50% inhibition of thestandard peptide at 11 nM) to the HLA-A2.1 molecule than G10₁₅₄ (1010nM) which contains an extra leucine at the C-terminus of G9₁₅₄, andcould sensitize T2 cells at lower concentrations than G10₁₅₄ (FIG. 8A).G9₂₀₉ also bound to HLA-A2.1 with higher affinity (84 nM) than G10₂₀₈(2080 nM) which contains an extra threonine at the N-terminus, and couldsensitize T2 cells at lower concentrations of peptide than G10₂₀₈ (FIG.8B). Thus, the 9-mer peptides were superior to the corresponding 10 merpeptides in the sensitization of T2 cells to CTL lysis, and they alsohad higher binding affinities to HLA-A2.1. This was also the case forthe identified MART-1 9 and 10 amino acid peptides (M9-2, M10-3, M10-4)(Example 2; Kawakami, Y., et al., (1994). J. Exp. Med. 180:347). Theresults of the peptide titration in the T2 cell lysis assay correlatedwith the results of the HLA-A2.1 binding affinity as measured by the invitro binding assay. The other gp100 epitopes, G9₂₈₀, G10-4, or G10-5had binding affinities for HLA-A2.1 with 50 inhibition at 95 nM, 483 nM,or 13 nM, respectively. The HLA-A2.1 binding affinities of thepreviously identified HLA-A2 restricted melanoma epitopes in MART-1(Example 2; Kawakami, Y., et al., (1994) J. Exp. Med. 180:347) andtyrosinase (Wolfel, T., (1994) Eur. J. Immunol. 24:759) were alsomeasured (M9-2(397 nM), M10-3(2272 nM), M10-4(5555 nM), T9₃₀₉(333 nM),T9₃₆₉ (40 nM)). Except for the 10mer peptides (G10₁₅₄, G10₂₀₈, GM-10-3,GM10-4), for which overlapping 9-mer epitopes (G9₁₅₄, G9₂₀₉, M9-2)existed, all melanoma epitopes had either high (G9₁₅₄, G10-5, T9₃₆₉) orintermediate (G9₂₀₉, G9₂₈₀, G10-4, M9-2, T9₁) binding affinities toHLA-A2.1.

Discussion

Multiple epitopes in the gp100 human melanoma antigen recognized by 4TIL which were associated with tumor regression when adoptivelytransferred to the autologous patients have been identified in thisstudy. Among the 5 epitopes described in this study, G9₁₅₄ or G10₁₅₄appeared to be the most commonly recognized, since these were recognizedby 3 of 4 gp100 reactive TIL derived from different patients. Althoughthe G9₂₈₀ peptide was reported to be recognized by all 5 CTL derivedfrom PBL of different patients (Cox, A. L., et al., (1994)). Science264:716), it was only recognized by 2 of 4 gp100 reactive TIL in thisstudy. This difference may be due to the sources of T-cells (TIL vs PBL)used.

It will be appreciated that the MART-1 peptide M9-2 may also bedesignated M9₂₇, the MART-1 peptide M10-3 may also be designated M10₂₆,and the MART-1 peptide M10-4 may also be designated M10₂₇. It will alsobe appreciated that the gp100 peptide G10-4 may also be designatedG10₄₅₇ and the gp100 peptide G10-5 may also be designated G10₄₇₆.

TABLE 11 Summary of antigen recognition by HLA-A2 restricted melanomaspecific TIL TIL 1200 620 660 1143 1074 1088 1235 1318 1363 1399Clinical* PR PR PR PR NR NR NR NR NR NR Response Antigen(epitope)gp100 + + + + − − − − − − (G9₁₅₄) (G9₂₀₉) (G9₂₈₀) (G9₂₈₀) (G10-4)(G9₁₅₄) (G9₁₅₄₎ (G10-5) MART-1 − + + + + + + + + + (M9-2) (M9-2) (M9-2)(M9-2) (M9-2) (M9-2) (M9-2) (M9-2) (M9-2) tyrosinase − − − − − − − − − −gp75 − − − − − − − − − − G9(10): gp100-9(10)-mer peptides, M9-2:MART-1₂₇₋₃₅ peptide Recognition of gp100 by TIL is significantly (p <0.001)correlated with clinical response for adoptive immunotherapy withHLA-A2 restricted TIL. *PR, partial response (>50% reduction in the sumof the product of perpendicular tumor diameters); NR, no response (<50%reduction)

TABLE 12 Recognition of gp100 peptides by TIL Target TIL cells Peptide(% specific lysis at E:T = 40:1) Exp. 1 620-1 620-2 660-1 1143 1200 1235624mel none 32 36 47 20 77 11 397mel none 2 3 0 0 0 0 T2 none 0 5 3 1 02 T2 M9-2 19 84 69 49 1 86 T2 G9₁₅₄ 0 21 4 0 100 0 T2 G10₁₅₄ 3 19 7 4 752 T2 G9₂₀₉ 45 21 0 3 0 0 T2 G10₂₀₈ 42 36 7 4 2 3 T2 G9₂₈₀ 2 7 43 11 0 0T2 G10-4 0 7 6 0 15 0 T2 G10-5 2 7 2 1 7 0 Exp. 2 620-1 620-2 660-2 11431200 1235 624mel none 60 65 74 49 82 18 397mel none 2 6 0 0 0 0 T2 none1 12 1 0 1 2 T2 M9-2 36 85 50 39 0 60 T2 G9₁₅₄ 5 27 32 1 78 5 T2 G10₁₅₄4 31 30 2 85 3 T2 G9₂₀₉ 22 74 5 4 1 3 T2 G10₂₀₈ 35 80 7 10 1 5 T2 G9₂₈₀*2 9 75 34 1 2 Exp. 3 660-3 1143 1200 1235 624mel none 52 15 66 40 397melnone 5 3 7 4 T2 none 7 3 7 4 T2 M9-2 50 62 4 94 T2 G9₂₈₀ 99 37 9 5 T2G10-4 0 0 50 0 T2 G10-5 14 2 6 5 Lysis by TIL of T2 cells preincubatedwith MART1 epitope, M9-2 (AAGIGILTV) and gp100 epitopes, G9₁₅₄(KTWGQYWQV), G10₁₅₄ (KTWGQYWQVL), G9₂₀₉(ITDQVPFSV), G10₂₀₈ (TITDQVPFSV),G9₂₈₀ (YLEPGPVTA), G10-4(LLDGTATLRL), G10-5 (VLYRYGSFSV) at 1 ug/ml (* 1ng/ml), was measured by 4 h-⁵¹Cr release assays. TIL620-1, -2 orTIL660-1, -2, -3 were grown from the same TIL which was administeredinto the autologous patient, but were separately cultured. 624mel,HLA-A2+ gp100+, MART-1+ melanoma cell line, 397mel, HLA-A2− melanomacell line. T2 cells, HLA-A2+ T cell-B-cell hybridoma. Bold:statistically significant lysis

TABLE 13 The relative binding affinity of the human melanoma epitopes toHLA-A2.1 Peptide Sequence 50% Ratio Protein (nM)^(a) Standard.^(b)inhibition to gp100 G9₁₅₄ KTWGQYWQV 11 0.45 G10₁₅₄ KTWGQYWQVL 1010 0.005G9₂₀₉ ITDQVPFSV 84 0.06 G10₂₀₈ TITDQVPFSV 2080 0.0024 G9₂₈₀ YLEPGPVTA 950.053 G10-4 LLDGTATLRL 483 0.01 G10-5 VLYRYGSFSV 13 0.38 MART-1 M9-2AAGIGILTV 395 0.013 M10-3 EAAGIGILTV 2272 0.0022 M10-4 AAGIGILTVI 55550.0009 Tyrosinase T9₁ MLLAVLYCL 333 0.015 T9₃₆₉ YMNGTMSQV 40 0.13^(a)Concentration of sample peptide required for 50% inhibition of thestandard radiolabeled peptide HBC18-27. ^(b)Ratio of the bindingaffinity of the sample peptide to that of the standard peptide (50%inhibition at 5 nM). Peptides are defined as high (50% inhibition at <50nM, ratio >0.1), intermediate (50-500 nM, ratio 0.1-0.01) and weak (>500nM, ratio <0.01) binding peptides.

EXAMPLE 5 Modification of melanoma Epitopes for Improvement ofImmunogenicity Material and Methods

Peptide synthesis and HLA-A2.1 binding assay. Peptides were synthesizedby a solid phase method using a multiple peptide synthesizer andpurified by HPLC, as previously described (Rivoltini, L et al. (1995)Journal of Immunology Volume 154:2257-2265). The relative binding ofpeptides to HLA-A2.1, based on the inhibition of binding of aradiolabeled standard peptide to detergent-solubilized MHC molecules,was performed as previously described (Rivoltini, L et al. (1995)Journal of Immunology Volume 154:2257-2265). Briefly, various doses ofthe test peptides (ranging from 100 μM to 1 nM) were coincubatedtogether with the 5 nM radiolabeled Hbc 18-27 (FLPSDYFPSV) peptide andHLA-A2.1 heavy chain and β2-microglobulin for 2 days at room temperaturein the presence of protease inhibitors. The percentage of MHC-boundradioactivity was determined by gel filtration and the 50% inhibitorydose was calculated for each peptide.

Induction of peptide specific CTL PBMC were separated from peripheralblood of HLA-A2⁺ melanoma patients and normal donors by centrifugationon Ficoll-Hypaque gradients and used as fresh or cryopreserved samples.Peptide specific CTL lines were generated as follows: at day 0, PBMCwere plated at a concentration of 1.5×10⁶/ml in 24-well plates (2ml/well) in Iscove's medium containing 10% human AB serum, L-glutamine,antibiotics (CM) and in the presence of 1 ug/ml peptide. Two days later,12 IU/ml interleukin 2 (IL-2) (Chiron Co., Emeryville, Calif.) wereadded to the cultures. Lymphocytes were then restimulated weekly asfollows: responder cells were harvested, washed once and replated in24-well plates at a concentration of 2.5×10⁵ cells/ml in CM. AutologousPBMC were thawed, washed twice in PBS, resuspended at 5-8×10⁶ cells/mlin CM and pulsed with 1 ug/ml peptide in 15-ml conical tubes (5 ml/tube)for 3 hours at 37° C. These PBMC (stimulators) were then irradiated at3000 rads, washed once in PBS and added to the responder cells atresponder:stimulator ratios ranging between 1:3 and 1:10. The next day,12 IU/ml IL-2 were added to the cultures. The activity of these CTL wastested by cytotoxicity assays after at least 2 rounds (14 days) ofpeptide stimulation. To generate CTL from TIL cultures, the dissociatedtumor suspension were cultured for 1-2 days in 10% FCS RPMI-1640 mediumto allow tumor cell adherence. The lymphocytes, recovered from thenon-adherent fraction, were used for the induction of peptide specificCTL as described above.

Assessment of antigen recognition by CTL. ⁵¹Cr release cytotoxic assayswere performed to detect the recognition of peptide and melanoma cellsby CTL. To analyze peptide recognition, T2 cell lines were preincubatedfor 2 h at 37° C. with 1 ug/ml peptide, washed and used as target cellsin a ⁵¹Cr release cytotoxic assay. The melanoma lines 624mel wasestablished in our laboratory (See Example 1).

In order to make more immunogenic peptides for induction ofanti-melanoma T-cells than natural melanoma epitopes, a variety ofpeptides in which at least 1 amino acid was changed based on consensusmotifs in peptides binding to a specific MHC Class I allele Falk, et al.(1991) Nature 351:290; Kubo et al. (1994) J. Immunol 152:3913; Parker,K. et al. (1992) Journal of Immunology 149:3580; Ruppert, J. et al.(1993) Cell 74:929-937) (Tables 14, 15, 16, and 17). Although most ofthe previously isolated viral epitopes and the naturally processedHLA-A2.1 binding peptides contained leucine or methionine at the 2ndmajor anchor position and valine at the last major anchor position(dominant anchor amino acids) and had high binding affinity to HLA-A2.1,the isolated MART-1 or gp100 melanoma epitopes contain non-dominantamino acid at major anchor position such as alanine (the 2nd position ofM9-2, the 9th position of G9-280) and threonine (the 2nd position ofG9-154 and G9-209). The M9-2, G9-209 and G9-280 are not high affinitybinders. By changing amino acid at the 1st, 2nd, 3rd or 9th positionswhich are important for HLA-A2 binding to the peptide, but lessimportant for recognition by T-cell receptors, artificial peptides whichcan bind to HLA-A2.1 with higher affinity and still be recognized bynatural epitope specific T-cells may be generated.

Among modified M9-2, G9-280, G9-209, G9-154 peptides, M9-2-2L, M9-2-1F,M9-2-3Y, G9-280-9V, G9-280-9L, G9-280-9I, G9-280-1F, G9-209-2L,G9-209-2M, G9-209-2I, G9-209-1F, G9-209-1Y, G9-209-1W2L, G9-209-1F2L,G9-209-1Y2L have higher binding affinity and were recognized by theoriginal melanoma reactive T-cells. (Tables 14, 15, 16 and 17) PBLstimulated with autologous PBMC pulsed with G9-154-2I, G9-209-1F2L, orG9-280-9V (Tables 18, 19 and 20) recognized and lysed not only theoriginal epitopes but also melanoma tumor cells (624mel) better than PBLstimulated with natural epitopes (G9-154, G9-209, G9-280).

These results demonstrated that modified peptides could be used forinduction of anti-tumor T-cells instead of natural epitopes. Otherpeptides which were not recognized by the particular T-cells used in ourstudy, but have higher binding affinity to HLA-A2.1 may induce adifferent set of T-cells capable of recognizing the original melanomaepitopes in in vitro or in vivo. These modified peptides may be used forinduction of anti-melanoma T-cells in vitro and immunization of patientsfor the treatment of patients with melanoma or for the prevention ofmelanoma.

TABLE 14 Modified MART-1 M9-2 peptides Binding Recognition affinity byM9-2 to HLA-A2.1 reactive Peptide Sequence (nM) T-cell M9-2 parentAAGIGILTV 1064 + M9-2-2L ALGIGILTV 10 + M9-2-2M AMGIGILTV 14 − M9-2-2IAIGIGILTV 77 − M9-2-1W WAGIGILTV 1351 + M9-2-1F FAGIGILTV 244 + M9-2-1YYAGIGILTV 136 − M9-2-3W AAWIGILTV 65 − M9-2-3F AAFIGILTV 67 − M9-2-3YAAYIGILTV 102 + M9-2-1K2L KLGIGILTV 14 − M9-2-1K2M KMGIGILTV 27 −M9-2-1K2I KIGIGILTV 94 − M9-2-1W2L WLGIGILTV 11 − M9-2-1F2L FLGIGILTV1.8 − M9-2-1Y2L YLGIGILTV 3.2 − M9-2-2L3W ALWIGILTV 5.5 − M9-2-2L3FALFIGILTV 1.4 − M9-2-2L3Y ALYIGILTV 3.7 −

TABLE 15 Modified gp100 G9-154 peptides Binding Recognition affinity byG9-154 to HLA-A2.1 reactive Peptide Sequence (nM) T-cell G9-154 parentKTWGQYWQV 5.7 + G9-154-2L KLWGQYWQV 2 + G9-154-2M KMWGQYWQV 6.5 +G9-154-2I KIWGQYWQV 3 + 09-154-1W WTWGQYWQV 60 − 09-154-1F FTWGQYWQV 1.6− G9-154-1Y YTWGQYWQV 2.5 − 09-154-1A ATWGQYWQV 5.2 + G9-154-1LLTWGQYWQV 3.4 + G9-154-3Y KTYGQYWQV 30 + 09-154-3F KTFGQYWQV 21 +G9-154-1A2L ALWGQYWQV 2.3 + G9-154-1L2L LLWGQYWQV 1.6 + G9-154-1W2LWLWGQYWQV 2.8 − 09-154-1F2L FLWGQYWQV 2.6 − G9-154-1Y2L YLWGQYWQV 1.7 −

TABLE 16 Modified gp100 G9-209 peptides Binding Recognition affinity byG9-209 to HLA-A2.1 reactive Peptide Sequence (nM)* T-cell G9-209 parentITDQVPFSV 172 + G9-209-2L ILDQVPFSV 3.3 + G9-209-2M IMDQVPFSV 19 +G9-209-2I IIDQVPFSV 40 + G9-209-1F FTDQVPFSV 61 + G9-209-1W WTDQVPFSV711 + G9-209-1Y YTDQVPFSV 85 + G9-209-3W ITWQVPFSV 34 − G9-209-3FITFQVPFSV 66 − G9-209-3Y ITYQVPFSV 33 − G9-209-3A ITAQVPFSV 95 −G9-209-3M ITMQVPFSV 40 − G9-209-3S ITSQVPFSV 649 − G9-209-2L3W ILWQVPFSV1.7 − G9-209-2L3F ILFQVPFSV 2 − G9-209-2L3Y ILYQVPFSV 5 − G9-209-2L3AILAQVPFSV 11 − G9-209-2L3M ILMQVPFSV 7.6 − G9-209-2L3S ILSQVPFSV 20 −G9-209-1W2L WLDQVPFSV 12 + G9-209-1F2L FLDQVPFSV 2.2 + G9-209-1Y2LYLDQVPFSV 2.3 + *a Concentration of sample peptide required for 50%inhibition of the standard radiolabeled peptide HBC18-27. Peptides aredefined as high (50% inhibition at <50 nM), intermediate (50-500 nM) andweak (>500 nM) binding peptides. (see ref gp100 epitope)

TABLE 17 Modified gp100 G9-280 peptides Binding Recognition affinity byG9-280 to HLA-A2.1 reactive Peptide Sequence (nM) T-cell G9-280 parentYLEPGPVTA 455 + G9-280-9V YLEPGPVTV 48 + G9-280-9L YLEPGPVTL 88 +G9-280-9I YLEPGPVTI 65 + G9-280-1F FLEPGPVTA 125 + G9-280-1W WLEPGPVTA833 + G9-280-3Y YLYPGPVTA 17 − G9-280-3W YLWPGPVTA 3.2 − G9-280-3FYLFPGPVTA 3.2 − G9-280-3M YLMPGPVTA 4.3 − G9-280-3S YLSPGPVTA 42 −G9-280-3A YLAPGPVTA 9.3 − G9-280-3M9V YLMPGPVTV 12 − G9-280-3S9VYLSPGPVTV 23 − G9-280-3A9V YLAPGPVTV 15 − G9-280-3Y9V YLYPGPVTV 8.9 −G9-280-3F9V YLFPGPVTV 5.8 − G9-280-3W9V YLWPGPVTV 7.4 −

TABLE 18 Induction of anti-melanoma CTL using modified G9-154 peptideEffector T-cells PBL stimulated PBL stimulated with G9-154 withG9-154-2I Target % specific lysis (E:T = 3D40:1) T2 11 1 T2+G9-154 14 37T2+G9-154-2I 8 38 624mel 5 23 ⁵¹Cr release assay was performed after 4times stimulation with autologous PBMC preincubated with peptides.

TABLE 19 Induction of anti-melanoma CTL using modified G9-209 peptideEffector T-cells PBL stimulated PBL stimulated with G9-209 withG9-209-1F2L Target % specific lysis (E:T = 3D40:1) T2 0 0 T2+G9-209 6 85T2+G9-209-1F2L 1 86 624mel 4 63 ⁵¹Cr release assay was performed after 4times stimulation with autologous PBMC preincubated with peptides.

TABLE 20 Induction of anti-melanoma CTL using modified G9-280 peptideEffectoe T-cells PBL stimulated PBL stimulated with G9-280 withG9-280-9V Target % specific lysis (E:T = 3D40:1) T2 3 0 T2+G9-280 11 87T2+G9-280-9V 8 58 624mel 11 71 ⁵¹Cr release assay was performed after 4times stimulation with autologous PBMC preincubated with peptides.

EXAMPLE 6 MART-1 vaccines as a Treatment for Melanoma in Mammals

MART-1 vaccines may be efficacious in treating mammals afflicted withmelanoma. For example, MART-1 vaccines may be administered toindividuals. Mammals can be immunized with the MART-1 proteins, peptidesor modified peptides described herein in ranges of about 1 mg-to toabout 100 mg. Alternatively mammals, preferably humans may be immunizedwith the MART-1 nucleic acid sequence inserted into a viral vector suchas vaccinia virus, adenovirus or fowl pox virus. A range of about 10⁶ toabout 10¹¹ viral particles carrying the MART-1 nucleic acid sequencescorresponding to immunogenic MART-1 peptides or modified peptides oranalogs therof, may be administered per mammal, preferably a human. Themammals will be monitored for antibodies to the immunogen or increase incytotoxic lymphocytes (CTL) recognizing the immunogen by conventionalmethods or alleviation of clinical signs and symptoms of the activedisease. Specific parameters to be assessed include production of immunecells that recognize the vaccine antigen or tumor regression. Suchvaccines may be administered either prophylactically or therapeutically.Mammals may also be immunized with the gp-100 nucleic acid sequenceinserted into a retroviral vector or GP-100 immunogenic peptides ormodified peptides or analogs thereof. Suggested dose ranges of theantigen in retroviruses that may be used are about 10⁶ to about 10¹¹viral particles per mammal, preferably a human. Response and efficacy ofthe retroviral vaccines will be assessed as described above.

EXAMPLE 7 Use of Lymphocytes Sensitized to Immunogenic Peptides Derivedfrom Melanoma Antigens for Therapeutically Treating Mammals Afflictedwith Melanoma

T-lymphocytes presensitized to the melanoma antigen may be effective intherapeutically treating mammals afflicted with melanoma. TheT-lymphocytes will be isolated from peripheral blood lymphocytes ortumor infiltrating lymphocytes and exposed in vitro to the MART-1protein or peptide. T-lymphocytes are isolated from peripheral blood ormelanoma tumor suspensions and cultured in vitro (Kawakami, Y. et al.(1988) J. Exp. Med. 168: 2183-2191). The T-lymphocytes are exposed tothe MART-1 peptide AAGIGILTV for a period of about to 1-16 hours at aconcentration of about 1 to about 10 mg/ml. T-lymphocytes exposed to theantigen will be administered to the mammal, preferably a human at about10⁹ to about 10¹² lymphocytes per mammal. Alternatively, theT-lymphocytes may be exposed to the modified MART-1 peptides. Thelymphocytes may be administered either intravenously, intraperitoneallyor intralesionally. This treatment may be administered concurrently withother therapeutic treatments such as cytokines, radiotherapy, surgicalexcision of melanoma lesions and chemotherapeutic drugs, adoptive Tlymphocyte therapy. Alternatively, the T-lymphocytes may be exposed tothe gp100 immunogenic peptides or modified immunogenic peptidesdescribed herein.

The present invention is not to be limited in scope by the nucleic acidsequences deposited, since the deposited embodiments is intended as asingle illustration of one aspect of the invention and any sequenceswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of thedependent claims.

1.-66. (canceled)
 67. An isolated host cell transformed or transfectedwith the recombinant expression vector comprising all or part of anucleic acid encoding a MART-1 protein (SEQ ID NO: 2) in a manner toallow expression of said protein encoded by said recombinant expressionvector.
 68. A vector comprising a nucleic acid encoding an immunogenicpeptide comprising 5-20 contiguous amino acids of gp100 (SEQ ID NO: 27)with at least one amino acid modification in the 5-20 contiguous aminoacids of SEQ ID NO: 27, wherein the immunogenic peptide reacts withT-lymphocytes.
 69. The vector of claim 68, wherein said modification anamino acid substitution.
 70. The vector of claim 69, wherein the aminoacid substitution is located at an amino acid position of theimmunogenic peptide selected from the group consisting of: (i) the firstposition, (ii) the second position, (iii) the third position, (iv) theninth position, (v) tenth position, and (vi) combinations of at leasttwo of (i)-(v).
 71. The vector of claim 70, wherein the immunogenicpeptide is selected from the group consisting of a group consisting ofKTWGOYWQV (SEQ ID NO: 46) with at least one amino acid modification inSEQ ID NO: 46, KTWGQYWQVL (SEQ ID NO: 47) with at least one amino acidmodification in SEQ ID NO: 47, ITDQVPFSV (SEQ ID NO: 48) with at leastone amino acid modification in SEQ ID NO: 48, TITDQVPFSV (SEQ ID NO: 49)with at least one amino acid modification in SEQ ID NO: 49, LLDGTATLRL(SEQ ID NO: 33) with at least one amino acid modification in SEQ ID NO:33, VLYRYGSFSV (SEQ ID NO: 34) with at least one amino acid modificationin SEQ ID NO: 34, VLKRCLLHL (SEQ ID NO: 36) with at least one amino acidmodification in SEQ ID NO: 36, ALDGGNKHFL (SEQ ID NO: 35) with at leastone amino acid modification in SEQ ID NO: 35, VLPSPACQLV (SEQ ID NO: 37)with at least one amino acid modification in SEQ ID NO: 37, YLEPGPVTA(SEQ ID NO: 40) with at least one amino acid modification in SEQ ID NO:40, SLADTNSLAV (SEQ ID NO: 38) with at least one amino acid modificationin SEQ ID NO: 38, SVSVSQLRA (SEQ ID NO: 39) with at least one amino acidmodification in SEQ ID NO: 39, and LNVSLADTN (SEQ ID NO: 41) with atleast one amino acid modification in SEQ ID NO:
 41. 72. A vectorcomprising an isolated nucleic acid encoding an immunogenic peptidecomprising an amino acid sequence of the formula selected from the groupconsisting of X₁X₂X₃ GQYWQX₄, X₁X₂X₃QVPFSX₄ and X₁X₂X₃PGPVTX₄, wherein:X₁ is any naturally occurring amino acid; X₂ is any hydrophobicaliphatic amino acid; X₃ is any naturally occurring amino acid; and X₄is any hydrophobic aliphatic amino acid.
 73. The vector of claim 72,wherein X₁ is selected from the group consisting of methionine, leucine,alanine, glycine, threonine, isoleucine, valine, tyrosine, serine,tryptophan, phenylalanine, lysine and aspartic acid.
 74. The vector ofclaim 72, wherein X₂ is selected from the group consisting ofmethionine, leucine, alanine, glycine, isoleucine, valine and threonine.75. The vector of claim 72, wherein X₃ is selected from the groupconsisting of methionine, leucine, alanine, glycine, threonine,isoleucine, tyrosine, valine, tryptophan, phenylalanine, serine, lysineand aspartic acid.
 76. The vector of claim 72, wherein X₄ is selectedfrom the group consisting of methionine, leucine, alanine, glycine,isoleucine, valine and threonine.
 77. The vector of claim 72, whereinthe immunogenic peptide is selected from the group consisting ofKTWGQYWQV (SEQ ID NO: 46), KLWGQYWQV (SEQ ID NO: 68), KMQGQYWQV (SEQ IDNO: 69), KIWGQYWQV (SEQ ID NO: 70), WTWGQYWQV (SEQ ID NO: 71), FTWGQYWQV(SEQ ID NO: 72), YTWGQYWQV (SEQ ID NO: 73), ATWGQYWQV (SEQ ID NO: 74),LTWGQYWQV (SEQ ID NO: 75), KTYGQYWQV (SEQ ID NO: 76), KTFGQYWQV (SEQ IDNO: 77), ALWGQYWQV (SEQ ID NO: 78), LLWGQYWQV (SEQ ID NO: 79), WLWGQYWQV(SEQ ID NO: 80), FLWGQYWQV (SEQ ID NO: 81), and YLWGQYWQV (SEQ ID NO:82).
 78. The vector of claim 72, wherein the immunogenic peptide isselected from the group consisting of ITDQVPFSV (SEQ ID NO: 48),ILDQVPFSV (SEQ ID NO: 83), IMDQVPFSV (SEQ ID NO: 84), IIDQVPFSV (SEQ IDNO: 85), FTDQVPFSV (SEQ ID NO: 86), WTDQVPFSV (SEQ ID NO: 87), YTDQVPFSV(SEQ ID NO: 88), ITWQVPFSV (SEQ ID NO: 89), ITFQVPFSV (SEQ ID NO: 90),ITYQVPFSV (SEQ ID NO: 91), ITAQVPFSV (SEQ ID NO: 92), ITMQVPFSV (SEQ IDNO: 93), ITSQVPFSV (SEQ ID NO: 94), ILWQVPFSV (SEQ ID NO: 95), ILFQVPFSV(SEQ ID NO: 96), ILYQVPFSV (SEQ ID NO: 97), ILAQVPFSV (SEQ ID NO: 98),ILMQVPFSV (SEQ ID NO: 99), ILSQVPFSV (SEQ ID NO: 100), WLDQVPFSV (SEQ IDNO: 101), FLDQVPFSV (SEQ ID NO: 102), and YLDQVPFSV (SEQ ID NO: 103).79. The vector of claim 72, wherein the immunogenic peptide is selectedfrom the group consisting of YLEPGPVTA (SEQ ID NO. 40), YLEPGPVTV (SEQID NO:104), YLEPGPVTL (SEQ ID NO:105), YLEPGPVTI (SEQ ID NO:106),FLEPGPVTA (SEQ ID NO: 107), WLEPGPVTA (SEQ ID NO: 108), YLYPGPVTA (SEQID NO: 109), YLWPGPVTA (SEQ ID NO: 110), YLFPGPVTA (SEQ ID NO: 111),YLMPGPVTA (SEQ ID NO: 112), YLSPGPVTA (SEQ ID NO: 113), YLAPGPVTA (SEQID NO: 114), YLMPGPVTV (SEQ ID NO: 115), YLSPGPVTV (SEQ ID NO: 116),YLAPGPVTV (SEQ ID NO: 117), YLYPGPVTV (SEQ ID NO: 118), YLFPGPVTV (SEQID NO: 119), and YLWPGPVTV (SEQ ID NO: 120).
 80. A host cell comprisingthe vector of claim
 68. 81. A host cell comprising the vector of claim72.